Addition Of Nifedipine Research Articles

JS-NG-10: A RARE CAUSE OF TREATMENT-RESISTANT HYPERTENSION

Objective: To present an educational case report. Design and method: In a 22-year old female arterial hypertension was diagnosed and she was treated to blood pressure target values with a combination of candesartan and lercanidipine. At the age of 27 years she planned a pregnancy, therefore antihypertensive treatment was switched to alpha-methyl-dopa and metoprolol. Blood pressure control was no longer achieved, the patient was symptomatic, mostly headaches. A diagnostic work-up for secondary causes of hypertension was performed. Results: There were no signs of kidney disease, renal artery stenosis was ruled out by color-coded duplex-ultrasound, plasma metanephrines were normal, sleep apnea syndrome and hypercortisolism were clinically very unlikely and not formally ruled out. No signs of hypertensive end-organ damage (left ventricular hypertrophy, microalbuminuria, carotid artery alterations) were detected. The only pathologic finding confirmed was a marked secondary hyperaldosteronism. Plasma renin concentration was 230 pg/ml, plasma aldosterone concentration 508 pg/ml, along with low normal serum potassium (3.5 to 4 mmol/l). Addition of nifedipine and low-dose HCT did not achieve blood pressure control, daytime ABPM mean was 144/102 mmHg, nighttime ABPM mean 127/86 mmHg. Because of suspected missed renal artery stenosis (fibromuscular dysplasia) an MR-angiography of the renal arteries was performed revealing normal renal arteries and normal adrenal glands but a left kidney tumor - not observed in the previous ultrasound examination but confirmed with contrast-enhanced ultrasound. Surgical removal of the tumor was performed. The histology was suggestive for a juxtaglomerular cell tumor (reninoma). After surgery, plasma aldosterone normalized to 57 pg/ml and plasma renin concentrations were low (3.4 pg/ml). Within 3 months after surgery, all antihypertensive medication could be discontinued and home blood pressure measurements averaged 119/78 mmHg. Soon thereafter the patient became pregnant and delivered a healthy girl. There were no pregnancy related complications and the patient remained normotensive. A juxtaglomerular cell tumor represents a rare curable cause of hypertension. The first report dates for 1967, until 2019 only about 100 cases were reported in the literature. It may be suspected that the diagnosis juxtaglomerular cell tumors is missed in many cases since these tumors are not easy to detect in standard diagnostic workup of hypertension and many antihypertensive drugs interfere with plasma renin concentrations.

The Dependence of Urinary Bladder Responses on Extracellular Calcium Varies Between Muscarinic, Histamine, 5-HT (Serotonin), Neurokinin, Prostaglandin, and Angiotensin Receptor Activation.

With many common bladder diseases arising due to abnormal contractions, a greater understanding of the receptor systems involved may aid the development of future treatments. The aim of this study was to identify any difference in the involvement of extracellular calcium (Ca2+) across prominent contractile-mediating receptors within cells lining the bladder. Strips of porcine urothelium and lamina propria were isolated from the urinary bladder dome and mounted in isolated tissue baths containing Krebs-bicarbonate solution, perfused with carbogen gas at 37°C. Tissue contractions, as well as changes to the frequency and amplitude of spontaneous activity were recorded after the addition of muscarinic, histamine, 5-hydroxytryptamine, neurokinin-A, prostaglandin E2, and angiotensin II receptor agonists in the absence and presence of 1 µM nifedipine or nominally zero Ca2+ solution. The absence of extracellular Ca2+ influx after immersion into nominally zero Ca2+ solution, or the addition of nifedipine, significantly inhibited the contractile responses (p < 0.05 for all) after stimulation with carbachol (1 µM), histamine (100 µM), 5-hydroxytryptamine (100 µM), neurokinin-A (300 nM), prostaglandin E2 (10 µM), and angiotensin II (100 nM). On average, Ca2+ influx from extracellular sources was responsible for between 20–50% of receptor-mediated contractions. This suggests that although the specific requirement of Ca2+ on contractile responses varies depending on the receptor, extracellular Ca2+ plays a key role in mediating G protein-coupled receptor contractions of the urothelium and lamina propria.

Estrogen Receptor Signaling and Intracellular Calcium Regulation in Human Airway Smooth Muscle

PurposeBronchial asthma is a chronic inflammation of the airways that is characterized by airway remodeling, hyperactive responsiveness and acute bronchoconstriction of airway smooth muscle (ASM) cells. Clinical findings suggest that there is a higher incidence of asthma in pre‐menopausal women and aging men, strongly indicating the role of sex steroids in modulating the airway tone. A major sex steroid, estrogen mediates its role through estrogen receptors (ER) ‐ ERα and ERβ. Our recent study demonstrated that these receptors are expressed in both male and female ASM cells and their expression is upregulated in the presence of inflammation/asthma. However, the role of ER signaling in asthma or genomic mechanisms in intracellular Ca2+ ([Ca2+]i ) regulation in human ASM are unknown. In this study, we evaluated the genomic effect of specific ERα and ERβ modulators on the [Ca2+]i in human ASM cells.MethodsHuman ASM cells were enzymatically dissociated from tissue obtained incidental to lung surgery from Mayo Clinic (IRB approved). Cells were plated onto 8‐well Lab‐Tek plates and grown to 70% confluence in a 5% CO2 humidified incubator. Cells were exposed to pro‐inflammatory cytokines TNFα (20ng/ml) or IL‐13 (50ng/ml) in the presence and absence of 1nM 17β‐estradiol (E2), 10nM PPT (ERα agonist) or WAY (ERβ agonist) for 24h. Cells were washed with HBSS and incubated with 3 μm Fluo‐3 for 1h for calcium imaging. The [Ca2+]i levels are measured from the intensity change observed following exposure to 10μM histamine under both physiological (2 mM) and zero extracellular calcium using Biotek LFX Fluorescence Imager.ResultsIn fluo‐3 loaded ASM cells, 24h exposure to WAY (and to a lesser extent PPT and E2) was effective in reducing [Ca2+]i levels. TNFα and IL‐13 significantly increased [Ca2+]i compared to vehicle. In the presence of TNFα or IL‐13, WAY significantly reduced [Ca2+]i in ASM cells, while PPT did not draw out any notable changes compared to TNFα or IL‐13 treatment alone. Addition of nifedipine (1μM, L‐type calcium channel blocker) resulted in a ~40% decrease in [Ca2+]i compared to control. This nifedipine blunting effect was similar in PPT treated group and to a higher extend to the WAY on [Ca2+]i responses to histamine, suggesting that ER effects were mediated via L‐type calcium channels. To verify the ER effect on sarcoplasmic reticulum (SR) Ca2+ reuptake, extracellular Ca2+ was removed by perfusion with zero Ca2+ HBSS, and Ca2+ influx was non‐specifically blocked with 1 mM LaCl3. Under these conditions, the [Ca2+]i responses were significantly reduced in the presence of WAY. Further, we examined effects of ER signaling on [Ca2+]i sequestration to SR (SERCA activity). In both control ASM cells as well as in those exposed to cytokines, WAY increased the rate of fall of [Ca2+]i responses to histamine, suggesting greater sequestration of [Ca2+]i.ConclusionsOverall, our data highlights that both ERα and ERβ specific agonist were able to reduce [Ca2+]i in the inflamed ASM via receptor‐mediated genomic action. Specifically, ERβ activation is more effective in the presence of inflammation.Support or Funding InformationSupported by NIH grants R01 HL123494 and R01 HL123494‐02S1 (Venkatachalem)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Molecular mechanism of Nifedipine inducing liver injury in children

Objective To establish the Nifedipine-induced liver cell damage model, and to investigate the cellular or molecular mechanism for children′s liver cell damage. Methods The HepG2 cells were utilized to establish liver cell damage models.The optimal concentration and the optimal pretreatment time of Nifedipine-induced liver cell injury were confirmed.Western blot and real-time PCR (RT-PCR) were used to check the alteration in proteins and mRNAs level of the liver function-associated classic markers, which contained alkaline phosphatase (ALP), aspartate amino transferase (AST), glutamyl transpeptidase (γ-GT) and alanine aminotransferase (ALT). Additionally, flow cytometry (FCM), colony formation assay (CFA) and cell wound healing assay (CWHA) were utilized to check the effect of Nifedipine on the cell cycle progression and proliferation of HepG2. Results (1)The optimal concentration of Nifedipine was 20 mg/L and the optimal treatment period was 21 days for liver damage.(2)Western blot: intracellular ALT protein content after Nifedipine group was less than that of control group, while the protein levels of AST, γ-GT and ALP in the culture medium after Nifedipine addition (3.55±0.05, 4.91±0.055, 3.51±0.05, 3.08±0.08) were higher than those of control group(0.96±0.02, 1.03±0.02, 1.00±0.05, 0.90±0.13), and the differences were all significant (t =-85.695, -117.582, -47.371, -33.260, all P<0.05). (3)The findings of RT-PCR showed that the mRNA levels of intracellular ALT, γ-GT and ALP in Nifedipine group (0.26±0.02, 0.05±0.04, 0.05±0.02) were lower than those of control group (1.13±0.21, 0.94±0.10, 1.03±0.06), and the differences were all significant (t=7.233, 127.436, 25.687, all P<0.05). However, the mRNAs levels in purified culture me-dium in Nifedipine group (5.95±0.05, 3.13±0.10, 3.32±0.08) were higher than those in control group (1.01±0.08, 1.00±0.05, 1.00±0.05), and the differences were all significant (t=-92.339, -31.250, -43.007, all P<0.05). (4)The portion of G0/G1 phase in Nifedipine group [(84.09±0.43)%] was more than that of control group[(30.93±0.32)%], which had statistical significance (t=173.084, P=0.000). (5)In contrast with control group, the colony formation of cells in Nifedipine group declined from (97.10±1.17)% to (38.56±1.51)% (t=92.088, P=0.000) and the migration rate of cells wound healing was (56.37±2.06)%, (25.00±1.71)% separately( t=20.285, P=0.000). Conclusion Nifedipine may promote children′s liver injury through regulating cell cycle related proteins. Key words: Nifedipine; HepG2; Liver function; Cell proliferation

Friedreich's ataxia induced pluripotent stem cell-derived cardiomyocytes display electrophysiological abnormalities and calcium handling deficiency.

We sought to identify the impacts of Friedreich's ataxia (FRDA) on cardiomyocytes. FRDA is an autosomal recessive degenerative condition with neuronal and non-neuronal manifestations, the latter including progressive cardiomyopathy of the left ventricle, the leading cause of death in FRDA. Little is known about the cellular pathogenesis of FRDA in cardiomyocytes. Induced pluripotent stem cells (iPSCs) were derived from three FRDA individuals with characterized GAA repeats. The cells were differentiated into cardiomyocytes to assess phenotypes. FRDA iPSC- cardiomyocytes retained low levels of FRATAXIN (FXN) mRNA and protein. Electrophysiology revealed an increased variation of FRDA- cardiomyocyte beating rates which was prevented by addition of nifedipine, suggestive of a calcium handling deficiency. Finally, calcium imaging was performed and we identified small amplitude, diastolic and systolic calcium transients confirming a deficiency in calcium handling. We defined a robust FRDA cardiac-specific electrophysiological profile in patient-derived iPSCs which could be used for high throughput compound screening. This cell-specific signature will contribute to the identification and screening of novel treatments for this life-threatening disease.

Abstract 34: STIM1 Increases Calcium Stores in the Sarcoplasmic Reticulum of Adult Feline Ventricular Myocytes

Background: STIM1 is a Sarcoplasmic Reticulum (SR) membrane resident protein implicated in sensing and maintaining SR Ca2+ levels. The role of STIM1 in the regulation of SR Ca2+ stores in the normal and diseased heart is not well described. Previous reports confirm that STIM1 is present at low levels in the healthy adult heart, but expression levels increase after cardiac injury. Objective: To determine if increased STIM1 expression after cardiac injury may be involved in the disturbed Ca2+ cycling present within diseased cardiomyocytes. Results: We used adenovirus to express either STIM1 or red fluorescent protein (RFP) in freshly isolated adult feline ventricular myocytes (AFMs). After 48 hours in culture, STIM1 induced cell death in 60% of myocytes versus only 5% in RFP controls. Addition of nifedipine rescued the cell death caused by STIM1, but block of transient receptor potential canonical (TRPC) channels was unable to improve viability. AFMs expressing STIM1 exhibited increased fractional shortening and Ca2+ transient amplitude, which was associated with increased SR load. Interestingly, high SR load levels caused by STIM1 were not reduced by nifedipine addition. We also found that baseline L-type channel current amplitude was significantly reduced by 20%, but no difference in current amplitude was found after Bay K8644 addition. Conclusions: In this study, we found that STIM1 caused Ca2+ overload leading to cell death. This process was L-type channel dependent, but TRPC channels were not involved. STIM1 also caused increased SR load at rest, which was not altered by nifedipine revealing that STIM1 mediated Ca2+ influx does not require L-type channel activity but Ca2+ influx through L-type channels is essential for STIM1 induced cellular death. Furthermore, STIM1 reduced L-type current activity but did not alter channel availability. These data show that after cardiac injury, STIM1 is likely to play a role in regulating cell survival as well as excitation-contraction coupling.

Genome Editing of Isogenic Human Induced Pluripotent Stem Cells Recapitulates Long QT Phenotype for Drug Testing

BackgroundHuman induced pluripotent stem cells (iPSCs) play an important role in disease modeling and drug testing. However, the current methods are time-consuming and lack an isogenic control. ObjectivesThis study sought to establish an efficient technology to generate human PSC-based disease models with isogenic control. MethodsThe ion channel genes KCNQ1 and KCNH2 with dominant negative mutations causing long QT syndrome types 1 and 2, respectively, were stably integrated into a safe harbor AAVS1 locus using zinc finger nuclease technology. ResultsPatch-clamp recording revealed that the edited iPSC-derived cardiomyocytes (iPSC-CMs) displayed characteristic long QT syndrome phenotype and significant prolongation of the action potential duration compared with the unedited control cells. Finally, addition of nifedipine (L-type calcium channel blocker) or pinacidil (KATP-channel opener) shortened the action potential duration of iPSC-CMs, confirming the validity of isogenic iPSC lines for drug testing in the future. ConclusionsOur study demonstrates that iPSC-CM-based disease models can be rapidly generated by overexpression of dominant negative gene mutants.

An evaluation of vardenafil as a calcium channel blocker in pulmonary artery in rats.

Objective:Vardenafil was reported to relax rat pulmonary artery through endothelium-dependent mechanisms. The aim of this in vitro study was to investigate other related mechanisms for this effect.Materials and Methods:Endothelium-intact and denuded artery rings were suspended in order to record isometric tension. In the rings with or without endothelium, the concentration-response curves for vardenafil were generated. In the rings without endothelium the contractile response induced by phenylephrine (Phe) or KCl was assessed in the presence or absence of vardenafil. In the last set of experiments, pulmonary artery rings were exposed to calcium-free isotonic depolarizing solution and the contractile response induced by the addition of calcium was evaluated in the presence or absence of vardenafil, nifedipine, verapamil or 1H-[1,2,4] oxadiazolo[4,3-a] quinoxalin-1-one (ODQ).Results:Vardenafil attenuated pulmonary artery contraction induced by phenylephrine in the presence and absence of endothelium. In addition, vardenafil attenuated both Phe or KCl-induced contraction but, it's effect on the KCl dose-response curve was more significant. Vardenafil also inhibited the contractile response induced by calcium in a dose-dependent manner. Addition of nifedipine or verapamil did not significantly alter this effect while ODQ incubation significantly inhibited vardenafil-induced relaxation.Conclusion:From these findings, it was proposed that vardenafil relaxed rat pulmonary artery through inhibiting calcium influx.

Single versus combination therapy in acute tocolysis: A prospective randomized controlled trial

This is a prospective controlled randomized trial conducted in 92 women with singleton pregnancies in preterm labor. The tocolytic efficacy and safety of combination atosiban and nifedipine was compared with that of the single agent, atosiban. Both lines of intervention was administered for 48 hours. Progression of labor was assessed by the frequency of uterine contractions and cervical changes. For statistical purpose, intent-to-treat (ITT) analysis was used throughout. Efficacy, as determined by the proportion of women in each group who did not deliver after therapy initiation, was comparable with no significant differences, at 48 hrs (91.5% vs. 91.1%) and at 7 days (90.7% vs. 85.7%) for the atosiban and the combination groups respectively. Safety was assessed by the numbers of adverse events. Maternal side effects were reported more in the combination group (34% vs. 64%; P = 0.006). Perinatal outcomes were similar between the groups. We conclude that the addition of nifedipine did not substantially improve the clinical outcomes beyond that were achieved with atosiban alone. Moreover, it has increased maternal side effects. Future research could focus on combination of other tocolytics.

Inhibitory effects of levetiracetam on the high-voltage-activated L-type Ca2+ channels in hippocampal CA3 neurons of spontaneously epileptic rat (SER)

Levetiracetam (LEV) is a widely used antiepileptic agent for partial refractory epilepsy in humans. LEV has unique antiepileptic effects in that it does not inhibit electroshock- or pentylenetetrazol-induced convulsion, but does inhibit seizures in kindling animal and spontaneously epileptic rat (SER: zi/zi, tm/tm) that shows both tonic convulsion and absence-like seizures. LEV also has unique characteristics in terms of its antiepileptic mechanism; it has no activity on Na⁺ and K⁺ channels or on glutamate and GABA(A) receptors. Recently, we found that LEV inhibits the depolarization shift and accompanying repetitive firing induced by mossy fiber stimulation in CA3 neurons of SER hippocampal slices. Therefore, this study was performed to determine whether LEV could inhibit the voltage-activated L-type Ca²⁺ current of hippocampal CA3 neurons obtained from SER and the non-epileptic Wistar rat. As previously reported, SER CA3 neurons were classified into type 1 and type 2 neurons. The application of LEV (100 μM) elevated the threshold for activation of the Ca²⁺ current, which was lowered in SER type 1 neurons and reduced the current size. Type 2 neurons of SER have a similar current-voltage relationship to Wistar rat neurons and the decay component of Ca²⁺ current during depolarization pulse in type 2 neurons was found to be smaller than that in Wistar rat neurons. LEV (100 μM) also reduced Ca²⁺ current in SER type 2 neurons. The effects of LEV were examined on such type 2 SER hippocampal CA3 neurons, compared with those on Wistar rat CA3 neurons. Application of LEV (10 μM) produced a significant decrease of amplitude of the Ca²⁺ current in SER neurons, although at this concentration of LEV there was no statistically significant decrease in the amplitude of Ca²⁺ current in Wistar rat neurons. Furthermore, LEV (100 nM-1 mM) reduced the Ca²⁺ current in a concentration-dependent manner in both SER and Wistar rat neurons, but the inhibition was much more potent in the former neurons than in the latter. Under the condition that the Ca²⁺ current had already been inhibited by LEV (10 μM), the addition of nifedipine (10 μM) did not cause further inhibition. Conversely, LEV had no effects on the current that had already been decreased by nifedipine (10 μM) given before LEV treatment (10 μM), indicating that LEV could act on the L-type Ca²⁺ channel. LEV elevated the threshold potential level for activation of the Ca²⁺ current and reduced the L-type Ca²⁺ current in type 1 neurons of SER, and the inhibitory action in type 2 neurons was much more potent than that in Wistar rat neurons, suggesting that these effects contribute, at least partly, to the antiepileptic action of LEV.

Activity-Dependent Transcriptional Regulation of M-Type K+ Channels by AKAP79/150-Mediated NFAT Actions

M-type K+ channels, encoded by the KCNQ2-5 family of genes, play key roles in the regulation of neuronal excitability; however, less is known about the mechanisms controlling their expression. Here we propose that neuronal stimulation induces elevated M-channel expression by activation of NFAT transcription factors in response to Ca2+/calcineurin (CaN)-mediated dephosphorylation, orchestrated by A-kinase-anchoring protein (AKAP) 79/150. We observed augmented mRNA for KCNQ2-3 and currents after stimulation of rat sympathetic neurons by either high-K+ or acetylcholine (ACh), by qRT-PCR and perforated-patch whole-cell clamp. Stimulation also elicited an increase in intracellular [Ca2+] and nuclear translocation of both endogenous and transfected GFP tagged-NFATc1-c2 from the cytoplasm to the nucelus. As evidence that this regulation of M channel expression is NFAT-mediated, exogenous expression of constitutively-active NFAT in sympathetic neurons increased tonic M-channel expression, which was not further increased by high-K+ stimulation. Moreover, this augmented transcription was suppressed by incubation of the neurons with the CaN inhibitor, cyclosporine A, and the membrane-permeable VIVIT inhibitory peptide, which competes with CaN for binding to NFATs. The involvement of AKAP79/150 in CaN/NFAT regulation of M-channel expression was also studied in sympathetic neurons isolated from AKAP150+/+ and AKAP150-/- mice. We found AKAP150-/- neurons to lack NFATc1 nuclear translocation and augmented expression of M channels after high-K+ stimulation, which was “rescued” by transfection of WT AKAP79 or a non-PKC binding AKAP79 mutant, but not AKAP79 mutants unable to bind CaN. Removal of external Ca2+ or addition of nifedipine during stimulation eliminated NFATc1 nuclear translocation and augmented M current, suggesting the role of L-type channels as the activity sensor. Thus, neuronal-activity regulates M-channel transcription, which in turn controls neuronal excitability. This activity-dependent transcriptional regulation of M channels involves a Ca2+-dependent NFAT signaling pathway, which requires AKAP79/150 targeting of CaN.

New Evidence of a Dihydropyridine-Activated Cationic Channel in the MDCK Cell Line

Newborn rat distal cells express an apical Ca²⁺ channel activated by dihydropyridine drugs. Similarly, in Madin-Darby canine kidney (MDCK) cells, nifedipine increased Ca²⁺_i in a concentration-dependent manner (IC₅₀ = 4 µM) in fura-2-loaded cells. Response to nifedipine was abolished by EGTA, suggesting that it depends on extracellular calcium. Ca²⁺ channel antagonist isradipine and agonist BayK8644 increased Ca²⁺_i indicating that this effect is related to the dihydropyridine group. Diltiazem (20 µM) and gadolinium (200 µM) decreased the nifedipine effect (62 and 43%, respectively). Lanthanum (100 µM) did not change the response. Valinomycin clamping of the membrane potential did not modify nifedipine-induced increment, indicating that it was unrelated to potassium fluxes. We performed whole cell clamp experiments in MDCK cells maintained at –50 mV with perfusion solution containing 10 mM CaCl₂. Nifedipine (20 µM) induced an increase in current (1.2 ± 0.3 nA), which was partially inhibited by Gd³⁺. No significant current was induced by nifedipine in the presence of 0.5 mM EGTA. To determine the effects of nifedipine on the membrane potential, we performed oxonol fluorescence experiments. The addition of nifedipine or Bay K8644 induced depolarization, highly dependent on external sodium. Nifedipine (20 µM) induced depolarization of 6.9 ± 0.8 mV (n = 21). EC₅₀ to nifedipine was in the 10 µM range. We conclude that MDCK cells exhibit a dihydropyridine-activated cationic channel.

Potentiating Effect of Daunorubicin on Vasocontractile Responses to KCl and BAY K 8644 in Rat Aorta

The effects of daunorubicin on vasoconstraction by several agonists have been investigated on isolated aortic strips from rats. Pretreatment of the strips with daunorubicin (17.7 microM) potentiated the contractile response to low concentrations of KCl or to BAY K 8644 but not to phenylephrine or clonidine. The maximal contractile response to KCl was not affected by the pretreatment while that to BAY K 8644 was increased. The potentiated response to KCl could be eliminated by addition of nifedipine (1 microM) or use of a calcium-free solution. The maximal contractile response to BAY K 8644 was greatly increased by partial depolarization with KCl (10 mM, final concn) in the control solution but only slightly increased by the partial depolarization in the solution with daunorubicin. These results suggest that daunorubicin facilitates activation of the voltage-dependent calcium channel and increases the contractile responses to KCl and BAY K 8644 in rat aorta.

Voltage-gated calcium channels are involved in the regulation of calcium oscillations in vascular smooth muscle cells from isolated porcine retinal arterioles

Disturbances in the regulation of retinal arteriolar tone are involved in the pathophysiology of major vision threatening diseases. Therefore, improving the treatment of these diseases requires knowledge about how the contraction of retinal arterioles is controlled. The present study hypothesized that in addition to calcium release from sarcoplasmic reticulum, calcium entry from the extracellular space is also involved in the generation of calcium oscillations as a basis for initiation of contraction in retinal arterioles. Isolated porcine retinal arterioles were mounted in a confocal microvascular myograph and loaded with the calcium sensitive fluorophore Oregon Green allowing simultaneous measurements of oscillations in the intracellular calcium concentration in individual smooth muscle cells and changes in the vascular tone. Recordings were made after pre-contraction with the thromboxane analogue U46619 in a low and a high ambient calcium concentration, and after subsequent blocking of voltage-gated calcium channels using nifedipine or the sarcoplasmic reticulum dependent calcium recruitment using ryanodine, caffeine, thapsigargin and cyclopiazonic acid. U46619 increased the number of oscillations in a low but not in physiological extracellular calcium concentration. In contrast to mesenteric arterioles, blocking of sarcoplasmic reticulum Ca 2+ channels with ryanodine was without effect on both retinal arteriolar tone and calcium oscillations. Conversely, blocking of the sarcoplasmic reticulum Ca 2+ pump with cyclopiazonic acid or thapsigargin significantly reduced calcium oscillations, but not arteriolar tone. At a physiological extracellular calcium concentration, addition of nifedipine, an L-type calcium channel blocker, reduced vascular tone but increased the number of cells displaying calcium oscillations. Fast oscillations in the intracellular calcium concentration in retinal smooth muscle cells depend on calcium transport by both the SR and the sarcolemma. However, both sites of calcium recruitment seem to have unique properties in porcine retinal arterioles as compared to other organs.

Sweet taste receptor expressed in pancreatic beta-cells activates the calcium and cyclic AMP signaling systems and stimulates insulin secretion.

BackgroundSweet taste receptor is expressed in the taste buds and enteroendocrine cells acting as a sugar sensor. We investigated the expression and function of the sweet taste receptor in MIN6 cells and mouse islets.Methodology/Principal FindingsThe expression of the sweet taste receptor was determined by RT–PCR and immunohistochemistry. Changes in cytoplasmic Ca2+ ([Ca2+]c) and cAMP ([cAMP]c) were monitored in MIN6 cells using fura-2 and Epac1-camps. Activation of protein kinase C was monitored by measuring translocation of MARCKS-GFP. Insulin was measured by radioimmunoassay. mRNA for T1R2, T1R3, and gustducin was expressed in MIN6 cells. In these cells, artificial sweeteners such as sucralose, succharin, and acesulfame-K increased insulin secretion and augmented secretion induced by glucose. Sucralose increased biphasic increase in [Ca2+]c. The second sustained phase was blocked by removal of extracellular calcium and addition of nifedipine. An inhibitor of inositol(1, 4, 5)-trisphophate receptor, 2-aminoethoxydiphenyl borate, blocked both phases of [Ca2+]c response. The effect of sucralose on [Ca2+]c was inhibited by gurmarin, an inhibitor of the sweet taste receptor, but not affected by a Gq inhibitor. Sucralose also induced sustained elevation of [cAMP]c, which was only partially inhibited by removal of extracellular calcium and nifedipine. Finally, mouse islets expressed T1R2 and T1R3, and artificial sweeteners stimulated insulin secretion.ConclusionsSweet taste receptor is expressed in β-cells, and activation of this receptor induces insulin secretion by Ca2+ and cAMP-dependent mechanisms.

Potassium Channel Opener Pinacidil Induces Relaxation of the Isolated Human Radial Artery

Taking into consideration that the search for drugs capable of modifying blood flow through human radial artery (RA) is warranted, the present study was designed to examine the vasodilatatory effects of the potassium channel opener, pinacidil on the RA and to define the contribution of different K+-channel subtypes in the endothelium-independent pinacidil action on this blood vessel. Pinacidil relaxed the RA rings with endothelium and without endothelium with comparable potency. N-nitro-L-arginine methyl ester (L-NAME) and methylene blue did not affect the pinacidil-induced vasorelaxation in rings with endothelium. In the rings without endothelium, the K+-channel blockers glibenclamide and tetraethylammonium (TEA) moderately antagonized the pinacidil-induced relaxation, while charybdotoxin and 4-aminopiridine did not. In endothelium-denuded rings, precontracted with 100 mM K+, the relaxant responses to pinacidil were highly significantly shifted to the right compared to those obtained in RA precontracted with phenylephrine, but pinacidil-induced maximal relaxation was not affected. Addition of nifedipine did not but addition of nifedipine and nickel (Na+-Ca2+ exchanger inhibitor) did cause a statistically significant rightward shift of the pinacidil concentration-relaxation curve, although the effect 0.1 mM pinacidil was preserved. Thus, pinacidil induces relaxation of the human RA in endothelium-independent manner, and glibenclamide- and TEA-sensitive vascular smooth muscle K+ channels are probably involved. Its ability to completely relax the RA precontracted with K+-rich solution suggests that pinacidil has additional K+ channel-independent mechanism(s) of action. It seems that stimulation of the forward mode of the Na+-Ca2+ exchanger plays a part in this K+ channel-independent effect of pinacidil.

Noradrenergic innervation of rabbit pancreatic ganglia

Sympathetic nerve stimulation indirectly regulates pancreatic endocrine and exocrine secretion, in part, through actions on the cholinergic parasympathetic innervation of the secretory tissues. Earlier work identified noradrenergic nerves in pancreatic ganglia and demonstrated the effects of exogenous norepinephrine (NE) on synaptic transmission but no quantitative studies of ganglionic NE content and release exist. Therefore, the distribution and density of catecholamine (CA)-containing nerves in rabbit pancreatic ganglia were studied using paraformaldehyde/glutaraldehyde (FAGLU) staining and HPLC analysis of CA concentrations. Neural release of [ 3H]NE was measured in ganglia isolated from the head/neck or body regions of the pancreas. CA-containing nerves densely innervated most ganglia (86%) from both regions, while neural and non-neural CA-containing cell bodies were rarely found. Ganglia from the head/neck region contained significantly higher concentrations of NE. Both 40 mM K + and veratridine evoked Ca 2+-dependent [ 3H]NE release and tetrodotoxin inhibited 80% of veratridine-stimulated release. ω-Conotoxin GVIA alone antagonized veratridine-stimulated release by 40% but the addition of nifedipine or ω-agatoxin IVA caused no further inhibition. There were no apparent regional differences in the Ca 2+-dependence or toxin-sensitivity of NE release. In conclusion, ganglia throughout the rabbit pancreas receive a dense, functional noradrenergic innervation and NE release is dependent upon N- but not P/Q- or L-type voltage-dependent Ca 2+ channels. These noradrenergic nerves may indirectly regulate pancreatic secretion through actions on ganglionic transmission.

Calcium mobilization is required for peroxynitrite-mediated enhancement of spontaneous transient outward currents in arteriolar smooth muscle cells

Transiently local release of Ca 2+ from the sarcoplasmic reticulum (SR) activates nearby Ca 2+-activated K + channels to produce spontaneous transient outward currents (STOCs) in smooth muscle cells. The purpose of the present study was to investigate the possible effect of peroxynitrite (ONOO −) on STOCs in mesenteric arteriolar smooth muscle cells (ASMCs) and decide whether Ca 2+ mobilization was involved in STOCs alteration by ONOO −. STOCs were recorded and characterized using the perforated whole-cell patch-clamp configuration. The results demonstrated that STOCs activity was greatly suppressed by removal of extracellular Ca 2+; by addition of nifedipine, a specific inhibitor of L-type voltage-gated Ca 2+ channels (VGCCs); or by addition of ryanodine, a SR ryanodine receptors (RyRs) blocker. In contrast, both caffeine, a RyR activator, and 2-aminoethoxydiphenylborate (2-APB), a membrane-permeable inositol 1,4,5-trisphosphate receptors, (IP 3R) antagonist, increased STOCs activity. 3-morpholinosydnonimine (SIN-1), an ONOO − donor, at concentrations of 20–200 μM, induced a dose-dependent enhancement of STOCs in ASMCs and led to conspicuous increases in STOCs frequency and amplitude, which were prevented by prior exposure to low external Ca 2+ (200 nM), ryanodine (10 μM), or nifedipine (10 μM). In contrast, caffeine (0.5 mM) did not further stimulate STOCs in ASMCs preincubated with SIN-1, and pretreatment with 2-APB (50 μM) had little effect on ONOO −-induced STOCs activation. These findings suggest that complex Ca 2+-mobilizing pathways, including external Ca 2+ influx through VGCCs activation and subsequent internal Ca 2+ release through RyRs but not IP 3Rs, are involved in ONOO −-mediated STOCs enhancement in ASMCs.

Estrogen induces nitric oxide production via activation of constitutive nitric oxide synthases in human neuroblastoma cells.

Although it is becoming increasingly evident that nitric oxide (NO) mediates some of estrogen's actions in the brain, the effects of estrogen on NO production through NO synthases (NOS) in neuronal cells have not yet been identified. Here we assessed changes in NO production induced by 17beta-estradiol (E2) in cells of neuronal origin using human SK-N-SH neuroblastoma cells, which we show express all three isoforms of NOS. Involvement of NOS isoforms in E2-induced NO production was examined using isoform-specific NOS inhibitors. E2 (10(-10)-10(-6) m) induced rapid increases in NO release and changes in endothelial NOS (eNOS) expression, which were blocked by ICI 182,780, an antagonist of estrogen receptors. Increased levels of NO release and NOS activity induced by E2 were blocked by N5-(1-Imino-3-butenyl)-L-ornithine, a neuronal NOS inhibitor, and N(5)-(1-Iminoethyl)-L-ornithine, an eNOS inhibitor, but not by 1400W, an inducible NOS inhibitor. These results demonstrate that E2-stimulated NO production occurs via estrogen receptor-mediated activation of the constitutive NOSs, neuronal NOS and eNOS. The E2-induced NO increase was abolished when extracellular Ca2+ was removed from the medium or after the addition of nifedipine, an L-type channel blocker, and was partially inhibited using 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid acetoxymethyl ester, an intracellular Ca2+ chelator. However, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid acetoxymethyl ester itself also caused an increase in NO release that was blocked by 1400W, suggesting that inducible NOS mediates this response. Together these data reveal that constitutive NOS activities are responsible for E2-induced NO production in neuroblastoma cells and that differential activation of NOS isoforms in these cells occurs in response to different treatments.

Pituitary adenylate cyclase activating polypeptide (PACAP) decreases neuronal somatostatin immunoreactivity in cultured guinea-pig parasympathetic cardiac ganglia

Postganglionic parasympathetic neurons in guinea-pig cardiac ganglia exhibit choline acetyltransferase (ChAT)-immunoreactivity, and a large fraction (60%) of the ChAT-positive cardiac neurons co-express somatostatin-immunoreactivity. This co-expression remained when the cardiac ganglia explants were maintained in culture for 72 h (40% somatostatin-immunoreactive). The guinea-pig cardiac ganglia neurons express the high affinity pituitary adenylate cyclase activating polypeptide (PACAP)-selective PAC 1 receptor, and treatment of the ganglia explants with 20 nM PACAP27 for 72 h to evaluate PACAP regulation of somatostatin expression revealed a dramatic 85% decrease in the number of somatostatin-IR neurons (6% somatostatin-IR neurons) compared with untreated control explant preparations. The decrease in percentage of somatostatin-IR neurons by PACAP27 was time- and concentration-dependent, and selective for PACAP27; PACAP38 and vasoactive intestinal polypeptide were less effective. PACAP6-38, a PACAP antagonist, eliminated the PACAP27-induced change in somatostatin positive neurons. The PACAP-mediated decrease in somatostatin-IR neurons was eliminated in calcium-deficient solutions and by the addition of nifedipine, indicating a requirement for calcium influx through L-type calcium channels. The addition of either the calmodulin inhibitor N-(4-aminobutyl)-1-naphthalenesulfonamide or the MEK inhibitor PD98059, also eliminated the PACAP27-induced decrease in somatostatin-IR cells. The PACAP27-mediated effect on somatostatin expression was not affected by inhibitors of protein kinase A or phospholipase C, but was reduced by the adenylyl cyclase inhibitor SQ22356, suggesting cAMP involvement. Semiquantitative and quantitative reverse transcription PCR prosomatostatin transcript measurements showed that cardiac ganglia prosomatostatin mRNA levels were not diminished by chronic PACAP27 exposure despite the dramatic decrement in somatostatin-expressing neurons. Neuronal peptide-IR content represents a balance between production and secretion. These results suggested that one of the primary effects of PACAP exposure may be enhanced levels of neuropeptide release that exceeded production levels, resulting in somatostatin depletion and a decrement in the number of identifiable somatostatin-expressing cardiac neurons.