Strips Of Mesenteric Arteries Research Articles

Molecular and cellular basis of praziquantel action in the cardiovascular system.

The anthelmintic drug praziquantel (PZQ) causes contraction of parasitic schistosomes as well as constriction of blood vessels within the mesenteric vasculature of the host where the adult blood flukes reside. The contractile action of PZQ on the vasculature is mediated by the activation of host serotonergic 5-HT2B receptors (5-HT2BRs). However, the molecular basis for PZQ interaction with these targets and the location of these 5-HT2B receptors in the vessel wall have not been experimentally defined. Evaluation of a PZQ docking pose within the 5-HT2BR orthosteric site, using both Ca2+ reporter and bioluminescence resonance energy transfer (BRET) assays, identified residues F3406.51 and F3416.52 (transmembrane helix 6, TM6) as well as L209EL2 (extracellular loop 2) as critical for PZQ-mediated agonist activity. A key determinant of PZQ selectivity for the 5-HT2B receptor over the 5-HT2A/2C receptors was determined by M2185.39 in transmembrane helix 5 (TM5) of the orthosteric site. Mutation of this residue to valine (M218V), as found in 5-HT2A and 5-HT2C, decreased PZQ agonist activity, whereas the reciprocal mutation (V215M) in 5-HT2C increased PZQ activity. Two-photon imaging in intact mesenteric arterial strips visualized PZQ-evoked Ca2+ transients within the smooth muscle cells of the vessel wall. PZQ also triggered cytoplasmic Ca2+ signals in arterial smooth muscle cells in primary culture that were isolated from mesenteric blood vessels. These data define the molecular basis for PZQ action on 5-HT2B receptors localized in vascular smooth muscle.

Agmatine suppresses peripheral sympathetic tone by inhibiting N-type Ca2+ channel activity via imidazoline I2 receptor activation

Agmatine, a putative endogenous ligand of imidazoline receptors, suppresses cardiovascular function by inhibiting peripheral sympathetic tone. However, the molecular identity of imidazoline receptor subtypes and its cellular mechanism underlying the agmatine-induced sympathetic suppression remains unknown. Meanwhile, N-type Ca2+ channels are important for the regulation of NA release in the peripheral sympathetic nervous system. Therefore, it is possible that agmatine suppresses NA release in peripheral sympathetic nerve terminals by inhibiting Ca2+ influx through N-type Ca2+ channels. We tested this hypothesis by investigating agmatine effect on electrical field stimulation (EFS)-evoked contraction and NA release in endothelium-denuded rat superior mesenteric arterial strips. We also investigated the effect of agmatine on the N-type Ca2+ current in superior cervical ganglion (SCG) neurons in rats. Our study demonstrates that agmatine suppresses peripheral sympathetic outflow via the imidazoline I2 receptor in rat mesenteric arteries. In addition, the agmatine-induced suppression of peripheral vascular sympathetic tone is mediated by modulating voltage-dependent N-type Ca2+ channels in sympathetic nerve terminals. These results suggest a potential cellular mechanism for the agmatine-induced suppression of peripheral sympathetic tone. Furthermore, they provide basic and theoretical information regarding the development of new agents to treat hypertension.

Suppression of peripheral sympathetic activity underlies protease-activated receptor 2-mediated hypotension.

Protease-activated receptor (PAR)-2 is expressed in endothelial cells and vascular smooth muscle cells. It plays a crucial role in regulating blood pressure via the modulation of peripheral vascular tone. Although some reports have suggested involvement of a neurogenic mechanism in PAR-2-induced hypotension, the accurate mechanism remains to be elucidated. To examine this possibility, we investigated the effect of PAR-2 activation on smooth muscle contraction evoked by electrical field stimulation (EFS) in the superior mesenteric artery. In the present study, PAR-2 agonists suppressed neurogenic contractions evoked by EFS in endothelium-denuded superior mesenteric arterial strips but did not affect contraction elicited by the external application of noradrenaline (NA). However, thrombin, a potent PAR-1 agonist, had no effect on EFS-evoked contraction. Additionally, ω-conotoxin GVIA (CgTx), a selective N-type Ca2+ channel (ICa-N) blocker, significantly inhibited EFS-evoked contraction, and this blockade almost completely occluded the suppression of EFS-evoked contraction by PAR-2 agonists. Finally, PAR-2 agonists suppressed the EFS-evoked overflow of NA in endothelium-denuded rat superior mesenteric arterial strips and this suppression was nearly completely occluded by ω-CgTx. These results suggest that activation of PAR-2 may suppress peripheral sympathetic outflow by modulating activity of ICa-N which are located in peripheral sympathetic nerve terminals, which results in PAR-2-induced hypotension.

Role of gap junction involved with endothelium-derived hyperpolarizing factor for the quercetin-induced vasodilatation in rat mesenteric artery

AimsModulation of vasodilating actions by quercetin, a kind of flavonoid, was investigated using rat mesenteric arterial ring strips. Main methodsRing strips (1mm) of rat mesenteric artery were used. The specimens were kept at 36.5°C in Krebs–Henseleit solution oxygenated with 95% O2 and 5% CO2. Key findingsQuercetin (0.1 to 100μM) dilated the contraction induced by norepinephrine (1μM) in a concentration-dependent manner. The quercetin-induced vasodilatation was almost resistant to both 100μM L-NG-nitro arginine methyl ester (L-NAME) and 100μM indomethacin. At 1mM tetraethylammonium (a KCa channel inhibitor) decreased the quercetin-induced vasodilatation, which was resistant to L-NAME and indomethacin, but not significantly. L-NAME- and indomethacin-resistant quercetin-induced vasodilatation was significantly attenuated by 100μM 18α- and 50μM 18β-glycyrrhetinic acids (gap junction inhibitors). Endothelium removal as well significantly attenuated the vasodilatation to the same extent. SignificanceThese results indicate that quercetin dilates the mesenteric artery via endothelium-dependent mechanisms, and the dilatation is mainly mediated by gap junctions closely involved with endothelium-derived hyperpolarizing factor (EDHF).

Propagation Mechanisms of Calcium Waves Arising During Arterial Vasomotion

Vasomotion consists in cyclic arterial diameter variations induced by synchronous contractions and relaxations of smooth muscle cells (SMCs) present in the arterial wall. These contractions have been shown to be due to an increase in the intracellular cytosolic calcium concentration. However, the arteries do not contract simultaneously on macroscopic distances and a propagation of the diameter variations can be observed. Our aim was to investigate this propagation. We stimulated endothelium denuded rat mesenteric arterial strips with the vasoconstrictor, phenylephrine (PE) to obtain vasomotion and observed that the contraction waves are linked to intercellular calcium waves through the SMCs. A velocity of about 100 μm/s was measured for the two kinds of waves which could propagate either from the distal to the proximal side of the artery or in the opposite direction. To investigate the calcium wave propagation mechanisms, we used a method allowing a PE stimulation of a small area of the strip. No calcium propagation could be induced by this local stimulation when the strip was in its resting state. However, if a low PE concentration was added to the whole strip, local PE stimulations induced calcium waves spreading over finite distances. The calcium wave velocity induced by local stimulation was identical to the velocity observed during vasomotion. This suggests that the propagation mechanisms are similar in the two cases. Using inhibitors of gap junctions and of voltage operated calcium channels, we showed that the locally induced calcium propagation likely depends on the propagation of the SMCs depolarization. Finally, from the experimental data gathered, it is possible to propose a model of the mechanisms underlying the propagation.

Intercellular calcium waves are associated with the propagation of vasomotion along arterial strips

Vasomotion consists of cyclic arterial diameter variations induced by synchronous contractions and relaxations of smooth muscle cells. However, the arteries do not contract simultaneously on macroscopic distances, and a propagation of the contraction can be observed. In the present study, our aim was to investigate this propagation. We stimulated endothelium-denuded rat mesenteric arterial strips with phenylephrine (PE) to obtain vasomotion and observed that the contraction waves are linked to intercellular calcium waves. A velocity of approximately 100 microm/s was measured for the two kinds of waves. To investigate the calcium wave propagation mechanisms, we used a method allowing a PE stimulation of a small area of the strip. No calcium propagation could be induced by this local stimulation when the strip was in its resting state. However, if a low PE concentration was added on the whole strip, local PE stimulations induced calcium waves, spreading over finite distances. The calcium wave velocity induced by local stimulation was identical to the velocity observed during vasomotion. This suggests that the propagation mechanisms are similar in the two cases. Using inhibitors of gap junctions and of voltage-operated calcium channels, we showed that the locally induced calcium propagation likely depends on the propagation of the smooth muscle cell depolarization. Finally, we proposed a model of the propagation mechanisms underlying these intercellular calcium waves.

Direct vasodilative effect of FK506 on porcine mesenteric artery in small bowel transplantation

Background Tacrolimus (FK506) is widely used as an immunosuppressive drug in small bowel transplantation. However, its precise effects on the vascular tone of the transplanted organ have not been studied. This study aimed to clarify the effects of FK506 on the porcine mesenteric artery. Methods The effects of FK506 on the changes in cytosolic Ca 2+ concentration ([Ca 2+]i) and force using fura-2 fluorometry were investigated in mesenteric arterial strips of the porcine small intestine. The effects of FK506 on the activity of voltage-dependent Ca 2+ channels and receptor-operated Ca 2+ channels using high K + (118 mmol/L K +) depolarization and thromboxane A 2 analog (U46619) stimulation were also examined. Results FK506 inhibited the force development induced by 118 mmol/L K + depolarization and 1 μmol/L U46619 stimulation in a concentration-dependent manner. The extent of inhibition of this contraction was greater than that of the K +-induced contraction, and its inhibitory potency was about 10-fold. FK506 (10 μmol/L) inhibited the increases in [Ca 2+]i (24.9% ± 7.4%) and the force development (52.0% ± 5.6%) induced by 1 μmol/L U46619, respectively. Conclusions FK506 induces arterial relaxation by decreasing [Ca 2+]i. Pretreatment of a graft with FK506 may reduce the risk of vasospasm, ischemia-reperfusion injury, and thrombosis in small bowel transplantation.

Does the Endothelium Abolish or Promote Arterial Vasomotion in Rat Mesenteric Arteries? Explanations for the Seemingly Contradictory Effects

Background and Aims: Vasomotion consists in cyclic oscillations of the arterial diameter induced by the synchronized activity of the smooth muscle cells. So far, contradictory results have emerged in the literature about the role of the endothelium in the onset and maintenance of vasomotion. Here our aim is to understand how the endothelium may either abolish or promote vasomotion. Methods and Results: We investigate rat mesenteric arterial strips stimulated with phenylephrine (PE). Our results show that the endothelium is not necessary for vasomotion. However, when the endothelium is removed, the PE concentration needed to induce vasomotion is lower and the rhythmic contractions occur for a narrower range of PE concentrations. We demonstrate that endothelium-derived relaxing products may either induce or abolish vasomotion. On the one hand, when the strip is tonically contracted in a nonoscillating state, an endothelium-derived relaxation may induce vasomotion. On the other hand, if the strip displays vasomotion with a medium mean contraction, a relaxation may induce a transition to a nonoscillating state with a small contraction. Conclusion: Our findings clarify the role of the endothelium on vasomotion and reconcile the seemingly contradictory observations reported in the literature.

COX-2 Up-regulation and vascular smooth muscle contractile hyperreactivity in spontaneous diabetic / mice

Abnormalities in vascular constriction and dilatation are associated with early diabetes and contribute to diabetic vascular complications. However, mechanisms underlying such vascular dysfunction remain to be fully elucidated. The current study tests the role of cyclooxygenase-2 (COX-2) in diabetic vascular smooth muscle dysfunction. Small mesenteric artery and aorta are isolated from type 2 diabetic db/db and control mice. Isometric contractions in response to serotonin, angiotensin II, phenylephrine and high potassium are determined in small spiral mesenteric arterial or aortic strips. COX-2 mRNA and protein levels are analyzed by using DNA microarray, real-time PCR and immunoblot. Contractions induced by serotonin, angiotensin II, phenylephrine and high potassium are significantly higher in endothelium-denuded smooth muscle strips isolated from db/db mice than in those isolated from control mice. The contractile hyperreactivity is observed in aortic and third-order branch small mesenteric arterial smooth muscle strips. DNA microarray, real-time PCR and immunoblot analysis show that compared with control mice, COX-2 mRNA and protein are significantly increased in db/db mice aortic smooth muscle. The COX-2 up-regulation is temporally associated with the development of diabetes mellitus and vascular smooth muscle contractile hyperreactivity. Inhibition of COX-2 with NS-398 or SC-58125 partially--but significantly--alleviates agonist-induced but not potassium-induced contractile hyperreactivity. In addition, serum isolated from db/db mice induces COX-2 expression and increases thromboxane A2 production in primary cultured vascular smooth muscle cells (VSMC). SQ-29548, a TP receptor antagonist, diminished the db/db mice vascular smooth muscle contractile hyperreactivity. COX-2 is up-regulated and contributes at least in part to the vascular smooth muscle contractile hyperreactivity in db/db mice.

Evidence for signaling via gap junctions from smooth muscle to endothelial cells in rat mesenteric arteries: possible implication of a second messenger

We investigated heterocellular communication in rat mesenteric arterial strips at the cellular level using confocal microscopy. To visualize Ca 2+ changes in different cell populations, smooth muscle cells (SMCs) were loaded with Fluo-4 and endothelial cells (ECs) with Fura red. SMC contraction was stimulated using high K + solution and Phenylephrine. Depending on vasoconstrictor concentration, intracellular Ca 2+ concentration ([Ca 2+] i) increased in a subpopulation of ECs 5–11 s after a [Ca 2+] i rise was observed in adjacent SMCs. This time interval suggests chemical coupling between SMCs and ECs via gap junctions. As potential chemical mediators we investigated Ca 2+ or inositol 1,4,5-trisphosphate (IP 3). First, phospholipase C inhibitor U-73122 was added to prevent IP 3 production in response to the [Ca 2+] i increase in SMCs. In high K + solution, all SMCs presented global and synchronous [Ca 2+] i increase, but no [Ca 2+] i variations were detected in ECs. Second, 2-aminoethoxydiphenylborate, an inhibitor of IP 3-induced Ca 2+ release, reduced the number of flashing ECs by 75 ± 3% ( n = 6). The number of flashing ECs was similarly reduced by adding the gap junction uncoupler palmitoleic acid. Thus, our results suggest a heterocellular communication through gap junctions from SMCs to ECs by diffusion, probably of IP 3.

Role of Ca 2+ in vascular smooth muscle contractions induced by Phoneutria nigriventer spider venom

Phoneutria nigriventer venom (PNV) contracts vascular tissues and increases arterial blood pressure. This study aimed to investigate the mechanisms involved on PNV-induced contractions of rabbit mesenteric and celiac arteries. Strips of mesenteric and celiac arteries were suspended in a cascade system and superfused with warmed and oxygenated Krebs solution. PNV was dialyzed in order to exclude the participation of biogenic amines in the contractions elicited by the venom. Noradrenaline (NA, 30–300 pmol), PNV (1–10 μg), Bay K-8644 (0.3–3 nmol) and KCl (10–100 μmol) dose-dependently contracted the preparations. Ca 2+-free solution reduced by 38 and 83% the PNV-induced contractions of mesenteric and celiac arteries, respectively. Subsequent infusion of EGTA (0.2 mM) suppressed the residual contractions. Nifedipine (1 μM) and verapamil (10 μM) abolished PNV- and Bay K-8644-evoked contractions, whereas those induced by NA were reduced to a lesser extent. Lanthanum chloride (0.2 mM) inhibited by 75–90% the mesenteric and celiac contractions mediated by PNV. Caffeine (2 mM) fully blocked contractions induced by NA (95% mean inhibition), but only partly reduced those induced by PNV (35% mean inhibition). Ryanodine (10 μM) inhibited by 50% the contractions evoked by NA, but had no effect on the PNV-induced contractions in both tissues. Our findings indicate that PNV contracts vascular smooth muscle mainly due to increased influx of Ca 2+ from extracellular sources.

Dissociation of coronary artery contractile hyperreactivity from hypertension

Background Both coronary artery contractile hyperreactivity and hypertension are associated with increased coronary artery disease. It is not known how coronary artery contractile hyperreactivity relates to hypertension. The current study tests the hypothesis that coronary artery contractile hyperreactivity can be dissociated from hypertension and therefore may contribute to the etiology of CAD independent of hypertension. Methods The contractile responses to 5-hydroxytryptamine (5-HT) and guanosine triphosphate (GTP) were determined in intact (nonpermeabilized) and α-toxin–permeabilized coronary artery strips and small mesenteric artery strips isolated from four rat strains: spontaneously hypertensive rats (SHR), Wistar-Kyoto rats (WKY), WKY-derived hypertensive rats (WKHT), and WKY-derived hyperactive rats (WKHA). Results The SHR and WKHT were hypertensive, whereas the WKY and WKHA subjects were normotensive. The coronary artery contractile reactivity to 5-HT was significantly higher in SHR when compared with WKY. The coronary artery contractile reactivity was of similar magnitude in WKHA and WKHT and was intermediate between that of SHR and WKY rats. GTP-induced Ca 2+ sensitization of contractions were significantly greater in SHR than in WKHT, WKHA, and WKY; in comparison, no significant difference was found among WKHT, WKHA, and WKY. In contrast to the findings in coronary arteries, there was no significant difference in 5-HT–induced contractions in small mesenteric artery strips isolated from SHR and WKY. Conclusions Coronary artery contractile reactivity to 5-HT does not correlate entirely with blood pressure (BP) values. In addition, G-protein–mediated Ca 2+ sensitization of contraction was increased and contributed to the coronary artery contractile hyperreactivity in SHR.

Heme oxygenase-mediated vasodilation involves vascular smooth muscle cell hyperpolarization.

Chronic hypoxia is associated with both blunted agonist-induced and myogenic vascular reactivity and is possibly due to an enhanced production of heme oxygenase (HO)-derived carbon monoxide (CO). However, the mechanism of endogenous CO-meditated vasodilation remains unclear. Isolated pressurized mesenteric arterioles from chronically hypoxic rats were administered the HO substrate heme-l-lysinate (HLL) in the presence or absence of iberiotoxin, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), ryanodine, or free radical spin traps (N-tert-butyl-alpha-phenylnitrone and 4,5-dihydroxy-1,3-benzenedisulfonic acid disodium salt). The effects of HLL administration on vascular smooth muscle (VSM) membrane potential were assessed in superior mesenteric artery strips in the presence and absence of zinc protoporphyrin IX or iberiotoxin. The vasodilatory responses to exogenous CO were assessed in the presence and absence of ODQ or iberiotoxin. HLL administration produced a dose-dependent vasodilatory response that was nearly eliminated in the presence of iberiotoxin. Neither ODQ, spin traps, nor ryanodine altered the vasodilatory response to HLL, although ODQ abolished the vasodilatory response to S-nitroso-N-acetyl-penicillamine. HLL administration produced a zinc protoporphyrin IX- and iberiotoxin-sensitive VSM cell hyperpolarization. Iberiotoxin and ODQ inhibited the vasodilatory response to exogenous CO. Thus the vasodilatory response to endogenous CO involves cGMP-independent activation of VSM large-conductance Ca2+-activated K+ channels and does not likely involve the formation of Ca2+ sparks emanating from ryanodine-sensitive stores.

Cytochrome p-450 epoxygenase products contribute to attenuated vasoconstriction after chronic hypoxia.

The systemic vasculature exhibits attenuated vasoconstriction following chronic hypoxia (CH) that is associated with endothelium-dependent vascular smooth muscle (VSM) cell hyperpolarization. We hypothesized that increased production of arachidonic acid metabolites such as the cyclooxygenase product prostacyclin or cytochrome p-450 (CYP) epoxygenase-derived epoxyeicosatrienoic acids (EETs) contributes to VSM cell hyperpolarization following CH. VSM cell resting membrane potential (Em) was measured in superior mesenteric artery strips isolated from rats with control barometric pressure (Pb, congruent with 630 Torr) and CH (Pb, 380 Torr for 48 h). VSM cell Em was normalized between groups following administration of the CYP inhibitors 17-octadecynoic acid and SKF-525A. VSM cell hyperpolarization after CH was not altered by cyclooxygenase inhibition, whereas the selective CYP2C9 inhibitor sulfaphenazole normalized VSM cell Em between groups. Iberiotoxin also normalized VSM cell Em, which suggests that large-conductance, Ca2+-activated K+ (BKCa) channel activity is increased after CH. Sulfaphenazole administration restored phenylephrine-induced and myogenic vasoconstriction and Ca2+ responses of mesenteric resistance arteries isolated from CH rats to control levels. Western blot experiments demonstrated that CYP2C9 protein levels were greater in mesenteric arteries from CH rats. In addition, 11,12-EET levels were elevated in endothelial cells from CH rats compared with controls. We conclude that enhanced CYP2C9 expression and 11,12-EET production following CH contributes to BKCa channel-dependent VSM cell hyperpolarization and attenuated vasoreactivity.

48-h Hypoxic exposure results in endothelium-dependent systemic vascular smooth muscle cell hyperpolarization.

Chronic hypoxia (CH) results in reduced sensitivity to vasoconstrictors in conscious rats that persists upon restoration of normoxia. We hypothesized that this effect is due to endothelium-dependent hyperpolarization of vascular smooth muscle (VSM) cells after CH. VSM cell resting membrane potential was determined for superior mesenteric artery strips isolated from CH rats (PB = 380 Torr for 48 h) and normoxic controls. VSM cells from CH rats studied under normoxia were hyperpolarized compared with controls. Resting vessel wall intracellular Ca(2+) concentration ([Ca(2+)](i)) and pressure-induced vasoconstriction were reduced in vessels isolated from CH rats compared with controls. Vasoconstriction and increases in vessel wall [Ca(2+)](i) in response to the alpha(1)-adrenergic agonist phenylephrine (PE) were also blunted in resistance arteries from CH rats. Removal of the endothelium normalized resting membrane potential, resting vessel wall [Ca(2+)](i), pressure-induced vasoconstrictor responses, and PE-induced constrictor and Ca(2+) responses between groups. Whereas VSM cell hyperpolarization persisted in the presence of nitric oxide synthase inhibition, heme oxygenase inhibition restored VSM cell resting membrane potential in vessels from CH rats to control levels. We conclude that endothelial derived CO accounts for persistent VSM cell hyperpolarization and vasoconstrictor hyporeactivity after CH.

Increased Ca2+ Buffering Function of Sarcoplasmic Reticulum in Small Mesenteric Arteries from Spontaneously Hypertensive Rats

We compared the Ca2+ buffering function of the superficial sarcoplasmic reticulum (SR) during rest and during contraction in endothelium-denuded strips of small mesenteric arteries from 13-week-old spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto rats (WKY). The addition of caffeine (1-20 mM) caused a transient contraction in both strains, and the contraction was significantly larger in SHR. When the SR Ca2+ buffering function was eliminated by cyclopiazonic acid (CPA; 10 microM) or thapsigargin (100 nM), both of which inhibit SR Ca2+-ATPase, or by ryanodine (10 microM), which depletes the SR Ca2+, there was a larger contraction in SHR than in WKY, suggesting that the Ca2+ buffering function of the SR during rest is more important in SHR than in WKY. Judging from the augmenting effects of these three agents on the contractile responses to Bay k 8644 (1-300 nM), an agonist of L-type Ca2+ channels, or norepinephrine (10(-9)-10(-4) M), an alpha-adrenoceptor agonist, the effects were significantly greater in SHR than in WKY. We conclude that 1) the Ca2+ influx during rest and during stimulation with Bay k 8644 or norepinephrine is strongly buffered by Ca2+ uptake into the superficial SR in the small mesenteric arteries from SHR and WKY; and 2) these Ca2+ buffering functions are increased in SHR because of the larger capacity of SR for Ca2+ storage.

Vascular Reactivity to Calcitonin Gene Related-Peptide Is Enhanced in Subtotal Nephrectomy-Salt Hypertension.

P44 We previously demonstrated that CGRP plays a counterregulatory role to attenuate the BP increase in subtotal nephrectomy-(SN)-salt-induced hypertension. This effect was observed in the absence of any increase in neuronal CGRP expression in this hypertensive model, suggesting that the mechanism of this compensatory vasodilator response is enhanced vascular reactivity to CGRP. To test this hypothesis, hypertension was induced in Sprague-Dawley rats (150-175 g, n=5) by SN and 1% saline drinking water. Controls (n=5) were sham operated and given tap water to drink. After eleven days, systolic BP was significantly higher in the SN compared to the control rats (220±7 vs 140±4 mmHg). Vascular reactivity was then examined in isolated endothelium-intact superior mesenteric arterial strips. Phenylephrine (10 -9 -3×5 -5 M)-induced contraction was not different between the two groups. CGRP (10 -11 -5×10 -8 M)-induced maximum relaxation in phenylephrine-contracted arteries was not altered between the SN and control arteries, however, CGRP was significantly more potent in the arteries from the SN group (EC50, 0.7±0.2 vs 2.5±0.8 nM). Phenylephrine-contracted arteries were also treated with forskolin (10 -9 -10 -6 M) since increased intracellular cAMP levels is a prominant mechanism linked to CGRP-mediated vasodilation. While no change in efficacy was observed, there was a marked decrease in response to forskolin in SN arteries (EC50, 327±146 vs 20±8 nM). To examine the BP response to exogenous CGRP in vivo, identical groups of SN and control rats (n=6/group) had i.v. (for CGRP administration) and arterial (for continous BP monitoring) catheters surgically placed and were studied in a concious, unrestrained state. Baseline mean BP was higher in the SN rats compared to the controls (173±5 vs 125±4 mmHg). Administration of CGRP (0.25μg/kg i.v.) resulted in a significantly greater decrease in mean BP in the SN rats compared to controls (-19±4 vs -9±1 mmHg). These data suggest that the counterregulatory effects of CGRP observed in SN-hypertension result, at least in part, from a cAMP-independent enhancement in vascular reactivity to this potent vasodilator.

Vascular Reactivity to Calcitonin Gene Related-Peptide Is Enhanced in Subtotal Nephrectomy-Salt Hypertension.

P44 We previously demonstrated that CGRP plays a counterregulatory role to attenuate the BP increase in subtotal nephrectomy-(SN)-salt-induced hypertension. This effect was observed in the absence of any increase in neuronal CGRP expression in this hypertensive model, suggesting that the mechanism of this compensatory vasodilator response is enhanced vascular reactivity to CGRP. To test this hypothesis, hypertension was induced in Sprague-Dawley rats (150-175 g, n=5) by SN and 1% saline drinking water. Controls (n=5) were sham operated and given tap water to drink. After eleven days, systolic BP was significantly higher in the SN compared to the control rats (220±7 vs 140±4 mmHg). Vascular reactivity was then examined in isolated endothelium-intact superior mesenteric arterial strips. Phenylephrine (10 -9 -3×5 -5 M)-induced contraction was not different between the two groups. CGRP (10 -11 -5×10 -8 M)-induced maximum relaxation in phenylephrine-contracted arteries was not altered between the SN and control arteries, however, CGRP was significantly more potent in the arteries from the SN group (EC50, 0.7±0.2 vs 2.5±0.8 nM). Phenylephrine-contracted arteries were also treated with forskolin (10 -9 -10 -6 M) since increased intracellular cAMP levels is a prominant mechanism linked to CGRP-mediated vasodilation. While no change in efficacy was observed, there was a marked decrease in response to forskolin in SN arteries (EC50, 327±146 vs 20±8 nM). To examine the BP response to exogenous CGRP in vivo, identical groups of SN and control rats (n=6/group) had i.v. (for CGRP administration) and arterial (for continous BP monitoring) catheters surgically placed and were studied in a concious, unrestrained state. Baseline mean BP was higher in the SN rats compared to the controls (173±5 vs 125±4 mmHg). Administration of CGRP (0.25μg/kg i.v.) resulted in a significantly greater decrease in mean BP in the SN rats compared to controls (-19±4 vs -9±1 mmHg). These data suggest that the counterregulatory effects of CGRP observed in SN-hypertension result, at least in part, from a cAMP-independent enhancement in vascular reactivity to this potent vasodilator.

Ca 2+ movement from leaky sarcoplasmic reticulum during contraction of rat arterial smooth muscles

To examine the Ca 2+ buffering function of the sarcoplasmic reticulum during arterial contraction, we studied Ca 2+ movement during stimulation with K + or norepinephrine in arteries with a leaky sarcoplasmic reticulum. Responses were compared in endothelium-denuded strips of femoral, mesenteric and carotid arteries of the rat. To make the sarcoplasmic reticulum leaky to Ca 2+, Ca 2+-induced Ca 2+ release channels of the sarcoplasmic reticulum were locked open by treatment with ryanodine plus caffeine. After ryanodine treatment, the contractile responses to K + (3–20 mM) were augmented when compared with control responses in femoral and mesenteric arteries, but were inhibited in the carotid artery. Similar results were obtained when the contractile responses to norepinephrine were determined. The inhibition by ryanodine of the K +- or norepinephrine-contractions seen in the carotid artery was reversed by pretreatment with cyclopiazonic acid (10 μM), an inhibitor of the sarcoplasmic reticulum Ca 2+-ATPase, but was not by charybdotoxin (100 nM), a blocker of Ca 2+-activated K + channels. We conclude that (1) after ryanodine treatment, Ca 2+ entering from the extracellular space during stimulation with K + or norepinephrine is first taken up into the leaky sarcoplasmic reticulum and then reaches the myofilaments in femoral and mesenteric arteries, while in the carotid artery, Ca 2+ leaked from the sarcoplasmic reticulum reaches mainly the plasma membrane from where it is extruded into the extracellular space, and (2) the different movement of Ca 2+ may be due to the relative location of the sarcoplasmic reticulum in the smooth muscle cell of each artery.

Ca 2+ uptake function of sarcoplasmic reticulum during contraction of rat arterial smooth muscles

To determine the Ca 2+ uptake function of the sarcoplasmic reticulum during contraction, the effects of cyclopiazonic acid or thapsigargin, agents that inhibit sarcoplasmic reticulum Ca 2+-ATPase, on the contractile responses to K + or norepinephrine were compared in endothelium-denuded strips of femoral, mesenteric and carotid arteries of the rat. The addition of K + (3–20 mM) to the strips caused a concentration-dependent contraction, and the sensitivity to K + was much higher in the carotid artery than in the other arteries. The preincubation of strips with cyclopiazonic acid (10 μM) or thapsigargin (100 nM) caused a leftward shift of the concentration–response curve for K +, and this effect was smaller in the carotid artery than in the other arteries. Inhibition of sarcoplasmic reticulum Ca 2+ uptake caused the sensitivity to K + to be similar in the three arteries. Similar results were obtained when the contractile responses to norepinephrine were determined. Cyclopiazonic acid itself induced similar transient contractions in the three arteries. The addition of caffeine (20 mM) caused a transient contraction that was smaller in the carotid artery than in the other arteries. We conclude that (1) the Ca 2+ influx during stimulation with K + or norepinephrine is buffered by the sarcoplasmic reticulum in femoral and mesenteric arteries, (2) this function is weak in the carotid artery, probably because the sarcoplasmic reticulum of this artery is almost filled with Ca 2+ in the resting state, and (3) the Ca 2+ uptake function of the sarcoplasmic reticulum during contraction is reflected by the contractile sensitivity in these arteries.