Phenylephrine-induced Contraction Research Articles

Isoflurane increases the activity of the vascular matrix metalloproteinase-2 in non-pregnant rats and increases the nitric oxide metabolites in pregnancy.

Surgeries that require general anesthesia occur in 1.5-2% of gestations. Isoflurane is frequently used because of its lower possibility of affecting fetal growth. Therefore, we examined the isoflurane anesthesia-induced effects on maternal hemodynamic and vascular changes. We hypothesized that isoflurane would enhance endothelium-dependent vasodilation as a consequence of increased nitric oxide and decreased metalloproteinases (MMPs). Female rats (n=28) were randomized into 4 groups (7 rats/group): conscious (non-anesthetized) non-pregnant group, non-pregnant anesthetized group, conscious pregnant group, and pregnant anesthetized group. Anesthesia was performed on the 20th pregnancy day, and hemodynamic parameters were monitored. Nitric oxide metabolites, gelatinolytic activity of MMP-2 and MMP-9, and the vascular function were assessed. Isoflurane caused no significant hemodynamic changes in pregnant compared with non-pregnant anesthetized group. Impaired acetylcholine-induced relaxations were observed only in conscious non-pregnant group (by approximately 62%) versus 81% for other groups. Phenylephrine-induced contractions were greater in endothelium-removed aorta segments of both pregnant groups (with or without isoflurane) compared with non-pregnant groups. Higher nitric oxide metabolites were observed in anesthetized pregnant in comparison with the other groups. Reductions in the 75 kDa activity and concomitant increases in 64 kDa MMP-2 isoforms were observed in aortas of pregnant anesthetized (or not) groups compared with conscious non-pregnant group. Isoflurane anesthesia shows stable effects on hemodynamic parameters and normal MMP-2 activation in pregnancy. Furthermore, there were increases in nitric oxide bioavailability, suggesting that isoflurane provides protective actions to the endothelium in pregnancy.

Small mesenteric artery passive mechanical properties in adult APOE3 & APOE4 Mice

Previous studies from our lab have demonstrated changes in vascular structure and function in posterior cerebral arteries and left common carotid artery (CA) in adult male and female mice expressing human-ApoE targeted replacement of APOE3 (B6.129P2-Apoetm2(APOE*3)MaeN8) and APOE4 (B6.129P2-Apoetm3(APOE*4)MaeN8) (Taconic Labs). Therefore, we hypothesized that similar changes may be observed in the systemic circulation. We isolated 4thor 5th-order mesenteric arteries from adult 6-month-old male and female mice expressing hAPOE3 (n=5) or hAPOE4 (n=4). Mesenteric artery segments (5-8 mm in length) were isolated and cannulated on an arteriograph to assess vascular mechanical properties; lumen diameter, wall thickness, wall:lumen ratio, distensibility, stress versus strain (SvS), compliance, incremental modulus of elasticity (Einc), and calculated pulse wave velocity under passive conditions (Ca2+-free Krebs plus diltiazem) at intraluminal pressures ranging from 10mmHg to 140mmHg. In addition, phenylephrine-induced contraction and potassium-induced contraction were assessed using Ca2+-containing Krebs. Data was analyzed using two-way ANOVA, Student’s t-test, and analysis of nonlinear fit. Values were considered statistically different at p<0.05. In our preliminary data, lumen diameter, distensibility, compliance, strain, and calculated pulse wave velocity were similar in APOE3 and APOE4 mice. Wall thickness and wall:lumen ratio were significantly greater in APOE4 compared to APOE3 mice. Wall stress and Einc were significantly less in APOE4 compared to APOE3 mice. Interestingly, the APOE4 mice exhibited a significantly lesser stress per given amount of strain compared to the APOE3 mice. Phenylephrine- and potassium-induced contractions were similar in APOE3 and APOE4 mice. This data suggests there are changes in passive mechanical properties in small mesenteric arteries from APOE4 mice, which resemble a hypertensive phenotype. ABRC/ADHS18-205211 (DME, CBJ), Arizona Alzheimer’s Consortium (funded by the Arizona Department of Health Services, Contract No. CTR040636) and matching funds from Midwestern University (DME), Biomedical Sciences Program (SM, IW, DME), Biomedical Sciences Start-up Funds (DME). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Potassium Ion Channel Modulation as a Potential Remedy for Cadmium-induced Hypertension

Objective: Hypertension remains a chief non-communicable lifestyle disease that is negatively associated with dietary cadmium (Cd). The exact mechanism of Cd toxicity and the mitigating role of potassium supplementation are not fully known, hence, this study aimed to investigate their effects on the aorta. Hypothesis: We hypothesized that potassium supplementation can modulate Cd-induced vascular dysfunction. Method: Six cohorts of control, Cd-exposed, and potassium supplemented male Sprague-Dawley rats were selected. Hypertension was induced using cadmium chloride (2.5 or 5 mg/kg b.w.). Blood pressure was measured twice weekly. Reactivity to pharmacological agents with and without potassium ion channel modulation was assessed in thoracic aortic rings after eight weeks, using an organ bath set-up. Data: Statistical analysis was done using one-way analysis of variance and the results were reported as mean ± standard error of the mean. The Games-Howell or Bonferroni post hoc test was used for multiple comparisons. Summary of Results: Cadmium significantly (p<0.05) elevated systolic, diastolic, mean arterial, and pulse pressures, reduced (p<0.001) phenylephrine-induced contraction mostly via BKCa channels, and augmented (p<0.05) sodium nitroprusside-induced relaxation via K-ATP, KV, and KIR channels, without affecting acetylcholine-induced relaxation. Potassium supplementation ameliorated these effects via KIR and BKCa channels. Conclusions: The results indicate that modulation of potassium ion channels in the vasculature might provide therapeutic management for environmentally-induced hypertension and should be further explored. Funding source: The Mona Campus Committee for Research and Publications and Graduate Awards, School for Graduate Studies and Research, University of the West Indies. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Chloroquine and cinchonine affect rat vascular smooth muscle tonus through calcium channels - in silico and in vitro approaches.

In the present study, two structurally similar alkaloids from trees of Cinchona genus, chloroquine and cinchonine, were examined for their vasorelaxant effects in amodel of phenylephrine-induced smooth muscle contractions. Potential mechanisms of action associated with endothelial vasorelaxant compounds, voltage-gated Ca2+ channels (LTCCs), and inositol triphosphate receptors were examined in isolated rat aortic rings. Also, an in silico approach was used to predict the activity of the two test compounds. Experimental results revealed that both chloroquine and cinchonine significantly decrease phenylephrine-induced smooth muscle contractions, although to adifferent extent. Evaluated mechanisms of action indicate that endothelium is not involved in the vasorelaxant action of the two tested alkaloids. On the other hand, voltage-gated Ca2+ channels were found to be the dominant way of action associated with the vasorelaxant action of chloroquine and cinchonine. Finally, IP3R is found to have only asmall impact on the observed activity of the tested compounds. Molecular docking studies predicted that chloroquine possesses asignificant activity toward asuitable model of LTCCs, while cinchonine does not. The results of the present study point to the fact that great caution should be paid while administering chloroquine to vulnerable patients, especially those with cardiovascular disorders (Tab. 3, Fig. 3, Ref. 28).

Adjuvant-induced arthritis promotes vascular hyporesponsiveness to phenylephrine through a nitric oxide-related mechanism.

Arthritis has important cardiovascular repercussions. Phenylephrine-induced vasoconstriction is impaired in rat aortas in the early phase of the adjuvant-induced arthritis (AIA), around the 15th day post-induction. Therefore, the present study aimed to verify the effects of AIA on hyporesponsiveness to phenylephrine in rat aortas. AIA was induced by intradermal injection of Mycobacterium tuberculosis (3.8 mg/dL) in the right hind paw of male Wistar rats (n=27). Functional experiments in isolated aortas were carried out 15 days after AIA induction. Morphometric and stereological analyses of the aortas were also performed 36 days after the induction of AIA. AIA did not promote structural modifications in the aortas at any of the time points studied. AIA reduced phenylephrine-induced contraction in endothelium-intact aortas, but not in endothelium-denuded aortas. However, AIA did not change KCl-induced contraction in either endothelium-intact or denuded aortas. L-NAME (non-selective NOS inhibitor), 1400W (selective iNOS inhibitor), and ODQ (guanylyl cyclase inhibitor) reversed AIA-induced hyporesponsiveness to phenylephrine in intact aortas. 7-NI (selective nNOS inhibitor) increased the contraction induced by phenylephrine in aortas from AIA rats. In summary, the hyporesponsiveness to phenylephrine induced by AIA was endothelium-dependent and mediated by iNOS-derived NO through activation of the NO-guanylyl cyclase pathway.

Elsholtzia ciliata Essential Oil Exhibits a Smooth Muscle Relaxant Effect.

A recent in vivo study in pigs demonstrated the hypotensive properties of essential oil extracted from the blossoming plant Elsholtzia ciliata. This study was designed to examine the effect of E. ciliata essential oil (EO) on smooth muscle contraction. Tension measurements were performed on prostate strips and intact aortic rings isolated from rats. Results showed that EO caused a concentration-dependent reduction in phenylephrine-induced contraction of both the prostate and aorta, with a more pronounced inhibitory effect in the prostate. The IC50 of EO for the prostate was 0.24 ± 0.03 µL/mL (n = 10) and for the aorta was 0.72 ± 0.11 µL/mL (n = 4, p < 0.05 vs. prostate). The chromatographic analysis identified elsholtzia ketone (10.64%) and dehydroelsholtzia ketone (86.23%) as the predominant compounds in the tested EO. Since both compounds feature a furan ring within their molecular structure, other furan ring-containing compounds, 2-acetylfuran (2AF) and 5-methylfurfural (5MFF), were examined. For the first time, our study demonstrated the relaxant effects of 2AF and 5MFF on smooth muscles. Further, results showed that EO, 2AF, and 5MFF altered the responsiveness of prostate smooth muscle cells to phenylephrine. Under control conditions, the EC50 of phenylephrine was 0.18 ± 0.03 µM (n = 5), while in the presence of EO, 2AF, or 5MFF, the EC50 values were 0.81 ± 0.3 µM (n = 5), 0.89 ± 0.11 µM (n = 5), and 0.69 ± 0.23 µM (n = 4), respectively, p < 0.05 vs. control. Analysis of the affinity of EO for α1-adrenergic receptors in the prostate suggested that EO at a certain range of concentrations has a competitive antagonistic effect on α1-adrenergic receptors. In conclusion, EO elicits a relaxant effect on smooth muscles which may be related to the inhibition of α1-adrenoreceptors.

Abstract 123: Partial Endothelial Cell In Vivo Reprogramming Reverses The Hypercontractility To Phenylephrine In Resistance Arteries Isolated From Males But Not From Females BPH/2J Hypertensive Mice

The Schlager hypertensive (BPH/2J) mouse strain is a genetic model of spontaneoushypertension. Endothelial dysfunction is a hallmark characteristic of hypertension,including endothelial phenotypic transition. We hypothesized that treating endothelialcells with the transcription factors Oct4, Sox2 and Klf4, will lead to partial reprogramming,reduce non-functional phenotype changes, and prevent vascular dysfunction. Mouseendothelial cells were transduced with a lentiviral vector containing VE-cadherin-Oct4-Sox2-Klf4-eGFP (LVTF), control lentivirus (LVCO), or phosphate buffer saline (PBS).After 72h, cell migration and proliferation, Ki67 marker, and the endothelial progenitormarkers (CD31, CD133 and CD34) were evaluated. Male or female BPH/2J mice (33-42-week-old) and their age-matched controls BPN/3J (normotensive) received a singleinjection of LVCO or LVTF (100μL) via the tail vein and were evaluated after 10 days.Systolic blood pressure (SBP) was measured directly by catheterization of the left carotidartery. Mesenteric resistance arteries were isolated and mounted on wire myographs toevaluate phenylephrine-induced contraction. Data were analyzed using One-way orTwo-way ANOVA, with Bonferroni correction ( p &lt;0.05). Lentivirus-transduced endothelialcells had eGFP fluorescence (LVCO: 45±2%, LVTF:19±1%, n=5), and LVTF increasedOct4 (LVTF: 5.6±0.7% vs. LVCO: 1.8±0.2% vs. PBS: 1.5±0.4%, n=5) and CD133 (LVTF:11.7±0.8% vs. LVCO: 7.6±0.3% vs. PBS: 6.5±0.5%, n=5), but not CD31, CD34 or Ki67.Cell migration was lower in LVTF (71±2%) than LVCO cells (90±2%, n=5). Male andfemale BPH/2J treated with LVCO had higher SBP (male: 103±3mmHg, female:95±4mmHg, n=2-3) compared to BPN/3J mice (male: 88±1mmHg, female: 80±4mmHg,n=2-6), and LVTF treatment tended to decrease SBP in male BPH/2J (86±2mmHg, n=2, p =0.0548) but not in female mice (90±4mmHg, n=3). Phenylephrine-induced contractionwas reduced in arteries from male BPH/2J treated with LVTF (6.77±0.90mN, n=3), butnot in female mice (11.01±1.45mN, n=3) compared to LVCO (male: 12.23±0.15mN;female: 10.77±0.92mN; n=2-3). These data reveal that partial endothelial cellreprogramming in vivo is a new strategy to prevent endothelial dysfunction in malehypertensive mice.1

CETP expression ameliorates endothelial function in female mice through estrogen receptor-α and endothelial nitric oxide synthase pathway.

Endothelial dysfunction is an early manifestation of atherosclerosis. The cholesteryl ester transfer protein (CETP) has been considered pro-atherogenic by reducing plasma HDL levels. However, CETP may exhibit cell- or tissue-specific effects. We have previously reported that male mice expressing the human CETP gene show impaired endothelium-mediated vascular relaxation associated with oxidative stress. Although sexual dimorphisms on the metabolic role of CETP has been proposed, possible sex differences in the vascular effects of CETP were not previously studied. Thus, here we investigated the endothelial function of female CETP transgenic mice as compared to non-transgenic controls (NTg). Aortas from CETP females presented preserved endothelium-dependent relaxation to acetylcholine and an endothelium-dependent reduction of phenylephrine-induced contraction. eNOS phosphorylation (Ser1177) and calcium-induced NO levels were enhanced, while reactive oxygen species (ROS) production and NOX2 and SOD2 expression were reduced in the CETP female aortas. Furthermore, CETP females exhibited increased aortic relaxation to 17β-estradiol (E2) and upregulation of heat shock protein 90 (HSP90) and caveolin-1, proteins that stabilize estrogen receptor (ER) in the caveolae. Indeed, CETP females showed an increased E2-induced relaxation in a manner sensitive to ERα and HSP90 inhibitors methylpiperidinopyrazole (MPP) and geldanamycin, respectively. MPP also impaired the relaxation response to acetylcholine in CETP but not in NTg females. Altogether, the study indicates that CETP expression ameliorates anti-contractile endothelial effect and relaxation to E2 in females. This was associated with less ROS production, and increased eNOS-NO and E2-ERα pathways. These results highlight the need for considering sex-specific effects of CETP on cardiovascular risk.

Dynamin-independent CaV1.2 and KCa1.1 channels regulation and vascular tone modulation by the mitochondrial fission inhibitors dynasore and dyngo-4a

A role for mitochondrial fission in vascular contraction has been proposed based on the vasorelaxant activity of the dynamin (and mitochondrial fission) inhibitors mdivi-1 and dynasore. However, mdivi-1 is capable to inhibit Ba2+ currents through CaV1.2 channels (IBa1.2), stimulate KCa1.1 channel currents (IKCa1.1), and modulate pathways key to the maintenance of vessel active tone in a dynamin-independent manner. Using a multidisciplinary approach, the present study demonstrates that dynasore, like mdivi-1, is a bi-functional vasodilator, blocking IBa1.2 and stimulating IKCa1.1 in rat tail artery myocytes, as well as promoting relaxation of rat aorta rings pre-contracted by either high K+ or phenylephrine. Conversely, its analogue dyngo-4a, though inhibiting mitochondrial fission triggered by phenylephrine and stimulating IKCa1.1, did not affect IBa1.2 but potentiated both high K+- and phenylephrine-induced contractions. Docking and molecular dynamics simulations identified the molecular basis supporting the different activity of dynasore and dyngo-4a at CaV1.2 and KCa1.1 channels. Mito-tempol only partially counteracted the effects of dynasore and dyngo-4a on phenylephrine-induced tone. In conclusion, the present data, along with previous observations (Ahmed et al., 2022) rise caution for the use of dynasore, mdivi-1, and dyngo-4a as tools to investigate the role of mitochondrial fission in vascular contraction: to this end, a selective dynamin inhibitor and/or a different experimental approach are needed.

Lipoprotein lipase underlies the vascular anti-contractile effect of perivascular adipose tissue (PVAT)

Physiologically, perivascular adipose tissue (PVAT) exerts an anti-contractile effect on the vasculature. This effect is mediated by the biosynthesis and liberation of a myriad of anti-contractile molecules (e.g., adipokines). However, this anti-contractile function generally is lost with aging and cardiovascular diseases such as hypertension and diabetes. Lipoprotein lipase (LPL) is an enzyme classically known for hydrolyzing triglycerides into free fatty acids and glycerol. However, it is unknown whether LPL underlies the anti-contractile effect of PVAT. Specifically, we hypothesized that by inhibiting lipoprotein lipase (LPL) activity with Atglistatin, we would abolish the anti-contractile effect of PVAT. To test this hypothesis, mesenteric resistance arteries (MRA), with and without PVAT, were isolated from 3-month-old male Wistar rats and mounted on a wire myograph. Phenylephrine concentration-response curves were performed with and without Atglistatin (10 -5 M). As expected, PVAT exerted a significant anti-contractile effect on phenylephrine-induced contraction [Emax (mN), -PVAT-vehicle: 18.1±3.0 vs. +PVAT-vehicle: 7.1±1.1, p&lt;0.05]. Incubation of MRA with intact PVAT with Atglistatin significantly increased contraction [Emax (mN), +PVAT-vehicle: 7.1±1.1vs. +PVAT-Atglistatin: 22.6±4.2, p&lt;0.05], suggesting that LPL is indeed important for the anti-contractile effect of PVAT. Crucially, no difference was observed with Atglistatin in MRA lacking PVAT [Emax (mN), -PVAT-vehicle: 18.1±3.0 vs. -PVAT-Atglistatin: 18.5±2.9, p&gt;0.05], indicating that Atglistatin was not increasing contraction via non-PVAT-dependent mechanisms. Overall, these data suggest that LPL is an important mechanism mediating the anti-contractile effect of PVAT and that reduced LPL activity could be contributing to pro-contractile PVAT in aging and cardiovascular diseases. NHLBI (R00HL151889), NIGMS (1P20GM103641 - Pilot Project), UofSC SOM (Startup funds) This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

TRPM8 channels do not regulate endothelium-dependent and -independent vascular reactivity in mice

Foremost among the ion channels that control vascular tone is the transient receptor potential (TRP) cation channels. The TRPM subfamily of the TRP channels includes eight members, expressed in various excitable and non-excitable cells and involved in diverse functions, such as sensing membrane potential, cold, taste, redox, osmolarity, and cell survival. Previous analysis of TRPM gene expression showed the highest expression of TRPM8 in intact blood vessels compared with other isoforms (TRPM1-7). Studies have also revealed that activating TRPM8 by menthol and icilin modulates endothelium-dependent and independent vascular reactivity. However, the non-selectivity of pharmacological TRPM8 modulators constitutes a significant limitation in the prior studies aimed at delineating TRPM8-dependent vasoregulation. In this study, we demonstrate that acetylcholine (ACH)- and sodium nitroprusside (SNP)-induced reduction in arterial pressure was unaltered in mice lacking TRPM8. We generated endothelial-specific knockout (KO) of TRPM8 (ecTRPM8-/-) by breeding TRPM8 floxed (TRPM8fl/fl) with Cdh5-Cre (VE-Cadherin-Cre) mice. Unlike global TRPM8 KO, ecTRPM8-/- mice were not resistant to cold temperatures. Phenylephrine-induced contraction of renal and mesenteric arteries isolated from TRPM8fl/fl and ecTRPM8-/- mice were similar. Also, ACH- and SNP-induced relaxation of phenylephrine-precontracted renal and mesenteric arteries from TRPM8fl/fl and ecTRPM8-/- mice were comparable. These findings suggest that TRPM8 channels do not regulate endothelium-dependent and -independent vasorelaxation in mice. Disclosure of Funding: NHLBI: R01 HL151735-01 and R01HL151735-S This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

TRPM8 channel activation induces relaxation in pudendal artery and corpus cavernosum: Can TRPM8 channel be a target to improve diabetic erectile dysfunction?

Erectile dysfunction (ED) is one of the most common and underestimated complications of diabetes. Previous studies have shown that TRPM8 activation triggers relaxation of internal pudendal arteries (IPA) with increased sensitivity in hypertensive rats. Despite the correlation between ED and diabetes, research on the role of TRPM8 channels in the IPA and corpus cavernosum (CC) in diabetes still needs to be elucidated. We hypothesized that preserved TRPM8 channel function in diabetic mice could be a therapeutic target for the treatment of ED. Male lean and db/db mice were euthanized, and the IPA were mounted on DMT wire myographs and CC were mounted on AVS organ bath for the measurement of isometric force. Concentration-response curves to icilin or menthol (TRPM8 agonists) were obtained. The CC TRPM8 channels expression was evaluated by western blot. Second harmonic generation was used to evaluate collagen deposition in IPA. Data are expressed as mean ± S.E.M of 3-5 mice. Non-linear regression curve was performed, and the maximum response (Emax) was analyzed using Student’s t test (p&lt;0.05). Similar TRPM8 channel expression was observed in the CC from lean and db/db mice. Menthol-induced relaxation of the CC was reduced in diabetic mice (Emax: 105.00 ± 5.00%) compared to lean mice (Emax: 139.44 ± 15.91%) . Relaxant effect induced by icilin and menthol was similar between IPA from db/db (Emax icilin: 95.56 ± 0.86% and Emax menthol: 93.88 ± 2.33%) and lean (Emax icilin: 97.05 ± 0.87% and Emax menthol: 101.10 ± 1.75%) mice. Interestingly, pre-incubation with icilin (10-4M) for 30 minutes decreased the potency of phenylephrine-induced contraction compared to untreated IPA rings in both db/db and lean mice. In the IPA, diabetes decreased the sensitivity to acetylcholine compared to lean. However, the relaxation induced by acetylcholine was not changed by pre-incubation with icilin. Diabetic mice (db/db) IPA tended to have a decrease in arterial collagen content compared to lean mice. Although menthol-induced relaxation of the CC was reduced in diabetic mice, icilin and menthol were able to promote similar relaxation in IPA from diabetic and lean mice. In this way, TRPM8 function was preserved in IPA from diabetic mice. Icilin reduced sensitivity to phenylephrine in IPA from both mice. These data suggest that activation of TRPM8 could be a therapeutic target for the treatment of ED. SMSNA, NIH (RO1DK132948) and FAPESB This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Lipoprotein lipase activation is a novel vascular anti-contractile mechanism that is lost in aging

Increasing incidence of cardiovascular events with age can be attributed to the natural deterioration in organ function, and manifests through a range of vascular phenotypes that include, endothelial dysfunction, hypercontractility, stiffening and remodeling, inflammation, and oxidative stress. Given the aging global population, the identification and translation of novel mechanisms and therapeutics will be essential to increase longevity and prevent premature death and disability. Physiologically, perivascular adipose tissue (PVAT) is known to impact vasculature by exerting an anti-contractile effect mediated by the biosynthesis and liberation of a myriad of anti-contractile molecules (e.g., adipokines). However, this anti-contractile function declines with age and in related diseases. Therefore, our study aimed to restore the anti-contractile effect of PVAT in arteries from aged animals. Specifically, we predicted that increasing lipoprotein lipase (LPL) activity would reinstate the anti-contractile effect of PVAT on aged arteries. To assess this hypothesis, mesenteric resistance arteries (MRA), with (+) and without (-) PVAT, were isolated from male Fisher 344 rats aged 3 (3M) or 16 months (16M) and mounted on a wire myograph. Phenylephrine (PE) concentration-response curves were measured in the presence or absence of LPL activator, NO-1886 (Ibrolipim, 10uM). As expected, PVAT exerted significant anti-contractile effect in MRA, regardless of age [PE 100uM (%), 3M (-PVAT): 112.4±13.3 vs. 3M (+PVAT): 27.6±28.5 {Figure 1.a.} and 16M (-PVAT): 115.4±23.1 vs. 16M (+PVAT): 35.0±34.1 {Figure 1.b.}]. Contrary to our hypothesis, NO-1886 had no effect on the anti-contractile effect of PVAT in 3M or 16M rats [PE 100uM (%), 3M (+PVAT +vehicle): 30.0±34.3 vs. 3M (+PVAT +NO-1886): 47.9±29.8 {Figure 1.c.} and 16M (+PVAT +vehicle): 48.6±42.3 vs. 16M (+PVAT +NO-1886): 60.0±36.9 {Figure 1.d.}]. Surprisingly, we observed an effect of NO-1886 in MRA in the absence of PVAT. Specifically, NO-1886 significantly decreased phenylephrine-induced contraction in 3M rats, but not 16M rats [PE 100uM (%), 3M (-PVAT +vehicle): 112.4±13.3 vs. 3M (-PVAT +NO-1886): 37.8±22.2 {Figure 1.e.} and 16M (-PVAT +vehicle): 115.4±23.1 vs. 16M (-PVAT +NO-1886): 103.1±23.1 {Figure 1.f.}]. Overall, this suggests that LPL has an important physiological role in controlling vascular contraction and this novel anti-contractile mechanism of LPL diminishes with biological age. Funding: NHLBI (R00HL151889), NIGMS (1P20GM103641 - Pilot Project), NIA (K01AG061263), UofSC SOM (Startup funds) This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

PD02-03 ARF1 GEF INHIBITOR GOLGICIDE A INHIBITS ADRENERGIC AND NON-ADRENERGIC PROSTATE SMOOTH MUSCLE CONTRACTION

You have accessJournal of UrologyCME1 Apr 2023PD02-03 ARF1 GEF INHIBITOR GOLGICIDE A INHIBITS ADRENERGIC AND NON-ADRENERGIC PROSTATE SMOOTH MUSCLE CONTRACTION Alexander Tamalunas, Amin Wendt, Ru Huang, Ruixiao Wang, Yuhan Liu, Beata Rutz, Anna Ciotkowska, Giuseppe Magistro, Christian Stief, and Martin Hennenberg Alexander TamalunasAlexander Tamalunas More articles by this author , Amin WendtAmin Wendt More articles by this author , Ru HuangRu Huang More articles by this author , Ruixiao WangRuixiao Wang More articles by this author , Yuhan LiuYuhan Liu More articles by this author , Beata RutzBeata Rutz More articles by this author , Anna CiotkowskaAnna Ciotkowska More articles by this author , Giuseppe MagistroGiuseppe Magistro More articles by this author , Christian StiefChristian Stief More articles by this author , and Martin HennenbergMartin Hennenberg More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000003219.03AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Prostate smooth muscle contraction is one of the main targets for medical treatment in lower urinary tract symptoms (LUTS) secondary to benign prostatic hyperplasia (BPH). Due to an unfavorable balance between side effects and limited efficacy, current medications cause high discontinuation rates. Agonist-induced smooth muscle contractions are mediated by activation of a1-adrenoceptors, endothelin and thromboxane, and their intracellular signaling pathways including monomeric GTPases. In general, monomeric GTPases are activated by GEFs, and deactivated by GAPs. Recently, we could show, that the small monomeric GTPase ARF6 is involved in prostate smooth muscle contraction. Here, we examined the effect of the ARF1 GEF inhibitor golgicide A on agonist-induced human prostate smooth muscle contraction. METHODS: Prostate tissues were obtained from patients undergoing radical prostatectomy for prostate cancer (n=30 patients). Contractility of prostate strips was then assessed in an organ bath. Data are presented as mean ±standard deviation, with Emax and EC50 values, while group p-value is given in the diagrams. RESULTS: Noradrenaline-induced adrenergic contractions were decreased by 47±12.1% with golgicide A (10µM) for concentrations of 1-100 µM (Figure 1A), by 30±6.6% for 1-100 µM of phenylephrine-induced contractions (Figure 1B), and by 61±10.1% for 1-100 µM of methoxamine-induced contractions (Figure 1C). Additionally, golgicide A inhibited non-adrenergic contraction by thromboxane A2 analogue U46619 by 82±19.6% for 1-300 µM of U46619-induced contractions (Figure 1D), endothelin-1 induced contraction by 53±7.0% for 1-30 µM of endothelin-1-induced contractions (Figure 1E), and EFS-induced neurogenic contractions by 76±16.6% for 2-32 Hz (Figure 1F). CONCLUSIONS: Golgicide A-sensitive GEFs are involved in regulation of human prostate smooth muscle contraction, by control of ARF1 GEF activity. Knowledge about GEFs and GAPs involved in smooth muscle contraction is still limited. However, increased understanding of prostate smooth muscle contraction may assist in developing new pharmaceutical targets for LUTS medication in the future. Source of Funding: None © 2023 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 209Issue Supplement 4April 2023Page: e69 Advertisement Copyright & Permissions© 2023 by American Urological Association Education and Research, Inc.MetricsAuthor Information Alexander Tamalunas More articles by this author Amin Wendt More articles by this author Ru Huang More articles by this author Ruixiao Wang More articles by this author Yuhan Liu More articles by this author Beata Rutz More articles by this author Anna Ciotkowska More articles by this author Giuseppe Magistro More articles by this author Christian Stief More articles by this author Martin Hennenberg More articles by this author Expand All Advertisement PDF downloadLoading ...

AMPK activation by metformin protects against pulmonary hypertension in rats and relaxes isolated human pulmonary artery

Pulmonary hypertension (PH) is associated with pulmonary vasoconstriction and endothelial dysfunction leading to impaired nitric oxide (NO) and prostacyclin (PGI2) pathways. Metformin, the first line treatment for type 2 diabetes and AMP-activated protein kinase (AMPK) activator, has been recently highlighted as a potential PH treatment. AMPK activation has been reported to improve endothelial function by enhancing endothelial NO synthase (eNOS) activity and to have relaxant effects in blood vessels. In this study, we examined the effect of metformin treatment on PH as well as on NO and PGI2 pathways in monocrotaline (MCT)-injected rats with established PH. Moreover, we investigated the anti-contractile effects of AMPK activators on endothelium-denuded human pulmonary arteries (HPA) from Non-PH and Group 3 PH patients (due to lung diseases and/or hypoxia). Furthermore, we explored the interaction between treprostinil and the AMPK/eNOS pathway. Our results showed that metformin protected against PH progression in MCT rats where it reduced the mean pulmonary artery pressure, pulmonary vascular remodeling and right ventricular hypertrophy and fibrosis compared to vehicle-treated MCT rats. The protective effects on rat lungs were mediated in part by increasing eNOS activity and protein kinase G-1 expression but not through the PGI2 pathway. In addition, incubation with AMPK activators reduced the phenylephrine-induced contraction of endothelium-denuded HPA from Non-PH and PH patients. Finally, treprostinil also augmented eNOS activity in HPA smooth muscle cells. In conclusion, we found that AMPK activation can enhance the NO pathway, attenuate vasoconstriction by direct effects on smooth muscles, and reverse established MCT-induced PH in rats.

Vascular Smooth Muscle TRPV4 (Transient Receptor Potential Vanilloid Family Member 4) Channels Regulate Vasoconstriction and Blood Pressure in Obesity.

Vascular endothelium and smooth muscle work together to keep the balance of vasomotor tone and jointly maintain vascular homeostasis. Ca2+-permeable ion channel TRPV4 (transient receptor potential vanilloid family member 4) in endothelial cells regulates endothelium-dependent vasodilation and contraction in various states. However, how vascular smooth muscle cell TRPV4 (TRPV4SMC) contributes to vascular function and blood pressure regulation in physiological and pathologically obese condition has not been fully studied. We generated smooth muscle TRPV4-deficient mice and developed diet-induced obese mice model and analyzed the role of TRPV4SMC in intracellular Ca2+ ([Ca2+]i) regulation and vasoconstriction. Vasomotor changes of mouse mesenteric artery were measured by wire, and pressure myography. [Ca2+]i were measured by fluo-4 staining. Blood pressure was recorded by telemetric device. Vascular TRPV4SMC played different roles in regulating vasomotor tone than endothelial TRPV4 due to their different features of [Ca2+]i regulation. Loss of TRPV4SMC attenuated U46619- and phenylephrine-induced contraction, suggesting its involvement in regulating vascular contractility. Mesenteric arteries from obese mice showed SMC hyperplasia, suggesting an increased level of TRPV4SMC. Loss of TRPV4SMC did not influence the development of obesity but protected mice from obesity-induced vasoconstriction and hypertension. In arteries deficient in SMC TRPV4, SMCs F-actin polymerization and RhoA dephosphorylation were attenuated under contractile stimuli. Moreover, SMC-dependent vasoconstriction was inhibited in human resistance arteries with TRPV4 inhibitor application. Our data identify TRPV4SMC as a regulator of vascular contraction in both physiological states and pathologically obese mice. TRPV4SMC contributes to the ontogeny of vasoconstriction and hypertension induced by TRPV4SMC over-expression in obese mice mesenteric artery.

Structurally diverse monoterpene indole alkaloids with vasorelaxant activities from the branches of Alstonia scholaris

Seven undescribed monoterpene indole alkaloids alstoscholarinines A‒G, along with nineteen known alkaloids, were isolated from the branches of Alstonia scholaris (L.) R. Br. The isolated alkaloids were classified into ten framework types. The structures of the undescribed alkaloids were elucidated by extensive spectroscopic analysis, ECD calculation, and single-crystal X-ray diffraction analysis. Alstoscholarinine A is an unreported and unusual monoterpene indole alkaloid incorporating three nitrogen atoms, characterized by a compact 6/5/6/6/6/5 hexacyclic system bearing a piperidine ring and a unique oxazolidine ring. Alstoscholarinine B represents the first naturally C-17 nor-isositsirikine-type alkaloid. Plausible biosynthetic pathways of alstoscholarinines A and B were proposed. All isolates were evaluated for their vasorelaxant activities against phenylephrine-induced contraction of rat mesenteric arteries. Among them, seven alkaloids showed significant vasorelaxant activities with EC50 values less than 10 μM. Importantly, the akuammicine-type alkaloids in this study showed much better vasorelaxant activities than other framework type alkaloids, indicating that this type of alkaloid may be a valuable source for the discovery of vasodilators. A preliminary structure-activity relationship for vasorelaxant activities of the isolated akuammicine-type alkaloids is also discussed.

Phenylephrine-Induced Contraction in Guinea Pig Thoracic Aorta Is Triggered by Stimulation of α1L-Adrenoceptors Functionally Coupled with Store-Operated Ca2+ Channels and Voltage-Dependent Ca2+ Channels.

We examined whether the α1L-adrenoceptor (AR), which shows low affinity (pA2 < 9) for prazosin (an α1-AR antagonist) and high affinity (pA2 ≈ 10) for tamsulosin/silodosin (α1A-AR antagonists), is involved in phenylephrine-induced contractions in the guinea pig (GP) thoracic aorta (TA). Intracellular signaling induced by α1L-AR activation was also examined by focusing on Ca2+ influx pathways. Tension changes of endothelium-denuded TAs were isometrically recorded and mRNA encoding α-ARs/Ca2+ channels and their related molecules were measured using RT-quantitative PCR. Phenylephrine-induced contractions were competitively inhibited by prazosin/tamsulosin, and their pA2 value were calculated to be 8.53/9.74, respectively. These contractions were also inhibited by silodosin concentration-dependently. However, the inhibition was not competitive fashion with the apparent pA2 value being 9.48. In contrast, phenylephrine-induced contractions were not substantially suppressed by L-765314 (an α1B-AR antagonist), BMY 7378 (an α1D-AR antagonist), yohimbine, and idazoxan (α2-AR antagonists). Phenylephrine-induced contractions were markedly inhibited by YM-254890 (a Gq protein inhibitor) or removal of extracellular Ca2+, and partially inhibited by verapamil (a voltage-dependent Ca2+ channel (VDCC) inhibitor). The residual contractions in the presence of verapamil were slightly inhibited by LOE 908 (a receptor-operated Ca2+ channel (ROCC) inhibitor) and strongly inhibited by SKF-96365 (a store-operated Ca2+ channel (SOCC) and ROCC inhibitor). Among the mRNA encoding α-ARs/SOCC-related molecules, α1A-AR (Adra1a)/Orai3, Orai1, and Stim2 were abundant in this tissue. In conclusion, phenylephrine-induced contractions in the GP TA can be triggered by stimulation of Gq protein-coupled α1L-AR, followed by activation of SOCCs and VDCCs.

Two new phenylpropanoids from the leaves of Rauvolfia vomitoria

ObjectiveStudy on the non-alkaloid chemical constituents with novel structure and their biological activities from the w-butanol fraction of the leaves of Rauvolfia vomitoria. MethodsThe w-butanol fraction of R. vomitoria was separated by silica gel, ODS, and Sephadex LH-20 chromatography column, as well as semi-preparative HPLC to obtain five compounds. Their structures were identified by extensive spectroscopic analysis, including HRESIMS, 1D and 2D NMR, and ECD analysis. All the isolated compounds were evaluated their AChE inhibitory activities by Ellman’s method with slight modification, their vasorelaxant activities against phenylephrine-induced contraction of rat mesenteric arteries, and their inhibitory activity of a-glucosidase employing pNPG as substrate. ResultsTwo new phenylpropanoids (1-2) along with three known compounds, methyl trans-3,4,5-trimethoxycinnamate (3), methyl cis-3,4,5-trimethoxycinnamate (4), and 3,4,5-trimethoxybenzoic acid methyl ester (5), were isolated from the leaves of R.vomitoria. The five isolated compounds did not show significant tested activity. ConclusionChemical investigation of the leaves of R. vomitoria led to the isolation and identification of two new phenylpropanoids (1–2), expanding the study of non-alkaloids in the genus of Rauvolfia and enriching the chemical diversity of this genus.

Upregulation of mechanosensitive channel Piezo1 involved in high shear stress-induced pulmonary hypertension

IntroductionPiezo1 is an important mechanosensitive channel implicated in vascular remodeling. However, the role of Piezo1 in different types of vascular cells during the development of pulmonary hypertension (PH) induced by high shear stress is largely unknown. Materials and methodsWe used a rat PH model established by left pulmonary artery ligation (LPAL, for 2–5 weeks), which mimics the high flow and hemodynamic stress, to study Piezo1 contribution to pulmonary vascular remodeling. ResultsRight ventricular systolic pressure (RVSP), a surrogate measure for pulmonary arterial systolic pressure, and right ventricular wall thickness, a measure for right ventricular hypertrophy, were significantly increased in LPAL rats compared with Sham-control (SHAM) rats. Rats in LPAL-5w groups developed remarkable pulmonary vascular remodeling, while phenylephrine-induced contraction and acetylcholine-induced relaxation were both significantly inhibited in these rats. Upregulation of Piezo1, in association with increase in cytosolic Ca2+ concentration ([Ca2+]cyt), was observed in pulmonary arterial smooth muscle cells (PASMCs) from LPAL-2w and LPAL-5w rats in comparison to the SHAM controls. Piezo1 upregulation in PASMCs from LPAL rats was directly related to Yes-associated protein (YAP)/ TEA domain transcription factor 4 (TEAD4). Piezo1 expression was also upregulated in the whole-lung tissue of LPAL rats. The endothelial upregulation of Piezo1 was related to transcriptional regulation by RELA (p65) and lung inflammation. ConclusionThe upregulation of Piezo1 in both PASMCs and ECs coordinates with each other via different cell signaling pathways to cause pulmonary vascular remodeling in LPAL-PH rats, providing novel insights into the cell-type specific pathogenic roles of Piezo1 in shear stress-associated experimental PH.