Segmental differences in the roles of rho-kinase and protein kinase C in mediating vasoconstriction.

Rho-kinase and protein kinase C (PKC) have each been reported to mediate vasoconstriction via calcium sensitization. However, the relative contributions of these two kinases to vascular contraction, and whether their roles vary between large and small arteries, are largely unknown. We therefore assessed the relative roles of rho-kinase and PKC in mediating vasoconstriction in arteries from three segments of the aortic and mesenteric vasculature. We studied contractile responses of rat isolated thoracic aorta (diameter approximately 2 mm), superior mesenteric artery (SMA; approximately 1.5 mm), and second order branches of the superior mesenteric artery (BMA; approximately 300 mum). The roles of rho-kinase and PKC in mediating contractile responses to phenylephrine, 9,11-dideoxy-9,11-methanoepoxy prostaglandin F(2alpha) (U46619), and KCl were assessed by using the rho-kinase inhibitor R-[+]-trans-N-[4-pyridyl]-4-[1-aminoethyl]-cycloheaxanecarboxamide (Y-27632) (1 and 10 muM) and the PKC inhibitor 3-[1-[3-(amidinothio)propyl-1H-indol-3-yl]-3-(1-methyl-1H-indol-3-yl) maleimide (Ro 31-8220) (5 muM). Contractile responses of aorta and SMA were reduced by either 1 or 10 muM Y-27632 (P < 0.05), whereas responses of BMA were reduced by 10 muM (P < 0.05) but not 1 muM Y-27632. In contrast, Ro 31-8220 partly reduced contractile responses in aorta and SMA (P < 0.05), but it abolished responses of BMA (P < 0.05). Cotreatment with Y-27632 and Ro 31-8220 markedly attenuated contractile responses to phenylephrine and KCl in all vessels, but it had only a moderate inhibitory effect on responses to U46619 in aorta and SMA. Thus, contractile responses of the larger arteries can involve both rho-kinase and PKC to varying degrees. Conversely, contractile responses of small mesenteric resistance arteries seem to be mediated exclusively by PKC, with no apparent role for rho-kinase.

Targeting Rho and Rho-kinase in the treatment of cardiovascular disease.

The small GTPase Rho and its downstream effector Rho-kinase contribute to agonist-induced vascular contraction via Ca2+ sensitization. Reasonably selective pharmacological inhibitors of these proteins have been developed and are now widely used experimentally to investigate the role of this signaling pathway in vascular function. Rho and Rho-kinase have attracted increasing clinical interest as a result of emerging evidence for their roles in the pathogenesis of several cardiovascular disorders, including hypertension, coronary and cerebral vasospasm, atherosclerosis and diabetes, and are now considered important future therapeutic targets. A major challenge lies in further developing selective inhibitors of this pathway beyond experimental use. Consideration should perhaps also be given to widening the application of existing clinical drugs now known to also interfere with Rho-Rho-kinase signaling.

Opposing roles of endothelial and smooth muscle phosphatidylinositol 3-kinase in vasoconstriction: effects of rho-kinase and hypertension.

Phosphatidylinositol 3-kinase (PI3K) can activate endothelial nitric oxide synthase (eNOS), leading to production of the vasodilator NO. In contrast, vascular smooth muscle (VSM) PI3K may partially mediate vascular contraction, particularly during hypertension. We tested whether endothelial and VSM PI3K may have opposing functional roles in regulating vascular contraction. Secondly, we tested whether the procontractile protein rho-kinase can suppress endothelial PI3K/eNOS activity in intact arteries, thus contributing to vasoconstriction by G protein-coupled receptor (GPCR) agonists. We studied contractile responses to the GPCR agonist phenylephrine, and the receptor-independent vasoconstrictor KCl, in aortic rings from Sprague-Dawley rats. In endothelium-intact rings, the PI3K inhibitor wortmannin (0.1 microM) markedly augmented responses to phenylephrine (P < 0.05) by approximately 50% but not to KCl. However, in endothelium-denuded or N(G)-nitro-L-arginine methyl ester (L-NAME) (100 microM)-treated rings, wortmannin reduced responses to phenylephrine and KCl (P < 0.05). Furthermore, the rhokinase inhibitor Y-27632 (R-[+]-trans-N-[4-pyridyl]-4-[1-aminoethyl]-cycloheaxanecarboxamide; 1 microM) abolished responses to phenylephrine, and this effect was partially reversed by wortmannin or L-NAME. The ability of wortmannin to oppose the effect of rho-kinase inhibition on contractions to phenylephrine was L-NAME-sensitive. In aortas from angiotensin II-induced hypertensive rats, relaxation to acetylcholine (10 microM) was impaired (P < 0.05), and vasoconstriction by phenylephrine was markedly enhanced and not further augmented by wortmannin. These data suggest that endothelial PI3K-induced NO production can modulate GPCR agonist-induced vascular contraction and that this effect is impaired in hypertension in association with endothelial dysfunction. In addition, endothelial rho-kinase may act to suppress PI3K activity and, hence, attenuate NO-mediated relaxation and augment GPCR-dependent contraction.

Evidence that estrogen suppresses rho-kinase function in the cerebral circulation in vivo.

BACKGROUND AND PURPOSE: Premenopausal women are less susceptible to cardiovascular diseases than men or postmenopausal women. Such disease states are often associated with increased vascular RhoA/Rho-kinase activity and decreased activity of nitric oxide (NO). This study tested whether female gender is associated with lower Rho-kinase activity or higher NO activity in cerebral arteries in vivo and whether estrogen contributes to any such gender differences. METHODS: Changes in basilar artery diameter were measured with the use of a cranial window preparation in anesthetized Sprague-Dawley rats. Some female rats were ovariectomized (OVX) and treated subcutaneously daily for 14 days with vehicle (dimethyl sulfoxide) or 17beta-estradiol. Vascular expression of RhoA or Rho-kinase was assessed by Western blotting. RESULTS: The Rho-kinase inhibitor Y-27632 was selectively approximately 3-fold more potent as a cerebral vasodilator in males versus females. Expression of total RhoA or Rho-kinase did not differ between males and females. In OVX rats, vasodilator responses to Y-27632 resembled responses in males. Treatment of OVX rats with 17beta-estradiol normalized the vasodilator effects of Y-27632 to be equivalent to responses in intact female controls. The NO synthase inhibitor N-nitro-l-arginine methyl ester caused approximately 50% greater constriction of the basilar artery in females versus males, but responses in OVX rats treated with either vehicle or 17beta-estradiol did not differ from those recorded in intact females. CONCLUSIONS: These data indicate that vascular Rho-kinase function is suppressed in females because of the effects of estrogen, whereas the higher NO activity in females is estrogen independent.

Chronic mevastatin modulates receptor-dependent vascular contraction in eNOS-deficient mice.

We tested the hypothesis that endothelial nitric oxide (NO) synthase (eNOS)-derived NO modulates rho-kinase-mediated vascular contraction. Because 3-hydroxy-3-methylglutaryl (HMG)-CoA-reductase inhibition can both upregulate eNOS expression and inhibit rhoA/rho-kinase function, a second hypothesis tested was that statin treatment modulates rho-kinase-mediated contraction and that this can occur independently of eNOS. Contractile responses to the receptor-dependent agonists serotonin and phenylephrine but not to the receptor-independent agent KCl were greater in aortic rings from eNOS-null (eNOS(-/-)) vs. wild-type (eNOS(+/+)) mice. Similarly enhanced responses were seen in eNOS(+/+) rings after acute NOS inhibition. The rho-kinase inhibitor Y-27632 abolished or profoundly attenuated responses to receptor agonists in both eNOS(+/+) and eNOS(-/-) rings, but responses in eNOS(+/+) were more sensitive to Y-27632. Mevastatin treatment (20 mg/kg sc per day, 14 days) reduced responses to serotonin and phenylephrine in female mice of both strains. KCl-induced contractions were slightly smaller in eNOS(+/+)-derived aortic rings only. Levels of plasma cholesterol, and aortic expression of rhoA and rho-kinase, did not differ between groups. Thus eNOS-derived NO suppresses rhoA/rho-kinase-mediated vascular contraction. Moreover, a similar suppressive effect on rho-kinase-mediated vasoconstriction by statin therapy occurs independently of effects on eNOS or plasma cholesterol.

Mechanisms in histamine-mediated secretion from adrenal chromaffin cells.

The great majority of the sustained secretory response of adrenal chromaffin cells to histamine is due to extracellular Ca(2+) influx through voltage-operated Ca(2+) channels (VOCCs). This is likely to be true also for other G protein-coupled receptor (GPCR) agonists that evoke catecholamine secretion from these cells. However, the mechanism by which these GPCRs activate VOCCs is not yet clear. A substantial amount of data have established that histamine acts on H(1) receptors to activate phospholipase C via a Pertussis toxin-resistant G protein, causing the production of inositol 1,4,5-trisphosphate and the mobilisation of store Ca(2+); however, the molecular events that lead to the activation of the VOCCs remain undefined. This review will summarise the known actions of histamine on cellular signalling pathways in adrenal chromaffin cells and relate them to the activation of extracellular Ca(2+) influx through voltage-operated channels, which evokes catecholamine secretion. These actions provide insight into how other GPCRs might activate Ca(2+) influx in many excitable and non-excitable cells.

Ions, channels, and receptors.

In this section eight presenters focus on three distinct aspects of chromaffin cell biology: first, the properties of neuronal nicotinic receptors; second, the shaping of the Ca(2+) signals that underlie chromaffin cell function; and third, the properties and expression of cell surface transporter proteins. Together these studies provide considerable new insight into the complexity of the signaling mechanisms that regulate the functional activity of the cell.

How does histamine evoke catecholamine secretion from bovine chromaffin cells?

Histamine-evoked catecholamine secretion from bovine chromaffin cells was studied. Secretion was not prevented by inhibitors of IP3 receptors, protein kinase C, or phospholipase C; depleting Ca(2+) stores; or omitting extracellular Na(+) or Cl(-). Patch clamp studies showed histamine inhibited an M current, causing depolarization and firing of action potentials.

Phospholipase C-mediated signalling is not required for histamine-induced catecholamine secretion from bovine chromaffin cells.

A possible role for signalling through phospholipase C in histamine-induced catecholamine secretion from bovine adrenal chromaffin cells has been investigated. Secretion evoked by histamine over 10 min was not prevented by inhibiting inositol-1,4,5-trisphosphate receptors with 2-APB, by blocking ryanodine receptors with a combination of ryanodine and caffeine, or by depleting intracellular Ca(2+) stores by pretreatment with thapsigargin. Inhibition of protein kinase C with Ro31-8220 also failed to reduce secretion. Inhibition of phospholipase C with ET-18-OCH(3) reduced both histamine- and K(+) -induced inositol phosphate responses by 70-80% without reducing their secretory responses. Stimulating phospholipase C with Pasteurella multocida toxin did not evoke secretion or enhance the secretory response to histamine. The secretory response to histamine was little affected by tetrodotoxin or by substituting extracellular Na(+) with N -methyl-d-glucamine(+) or choline(+), or by substituting external Cl(-) with nitrate(-). Blocking various K(+) channels with apamin, charybdotoxin, Ba(2+), tetraethylammonium, 4-aminopyridine, tertiapin or glibenclamide failed to reduce the ability of histamine to evoke secretion. These results indicate that histamine evokes secretion by a mechanism that does not require inositol-1,4,5-trisphosphate-mediated mobilization of stored Ca(2+), diacylglycerol-mediated activation of protein kinase C, or activation of phospholipase C. The results are consistent with histamine acting by depolarizing chromaffin cells through a phospholipase C-independent mechanism.

Histamine promotes excitability in bovine adrenal chromaffin cells by inhibiting an M-current.

The current study has investigated the electrophysiological responses evoked by histamine in bovine adrenal chromaffin cells using perforated-patch techniques. Histamine caused a transient hyperpolarization followed by a sustained depolarization of 7.2 +/- 1.4 mV associated with an increase in spontaneous action potential frequency. The hyperpolarization was abolished after depleting intracellular Ca(2+) stores with thapsigargin (100 nM), and was reduced by 40 % with apamin (100 nM). Membrane resistance increased by about 60 % during the histamine-induced depolarization suggesting inhibition of a K(+) channel. An inward current relaxation, typical of an M-current, was observed in response to negative voltage steps from a holding potential of -30 mV. This current reversed at -81.6 +/- 1.8 mV and was abolished by the M-channel inhibitor linopirdine (100 microM). During application of histamine, the amplitude of M-currents recorded at a time corresponding with the sustained depolarization was reduced by 40 %. No inward current rectification was observed in the range -150 to -70 mV, and glibenclamide (10 microM) had no effect on either resting membrane potential or the response to histamine. The results show that an M-current is present in bovine chromaffin cells and that this current is inhibited during sustained application of histamine, resulting in membrane depolarization and increased discharge of action potentials. These results demonstrate for the first time a possible mechanism coupling histamine receptors to activation of voltage-operated Ca(2+) channels in these cells.

Caffeine stimulates Ca(2+) entry through store-operated channels to activate tyrosine hydroxylase in bovine chromaffin cells.

The ability of caffeine-induced store Ca(2+) mobilization to activate tyrosine hydroxylase was studied in bovine adrenal chromaffin cells. Caffeine increased tyrosine hydroxylase activity over 10 min with an EC(50) of 3 mm and maximum effect at 20 mm. The maximum response to caffeine was substantial, being almost one third that of the strongest agonists acetylcholine and PACAP-27, about half that for K(+) and similar to that for histamine. In contrast, catecholamine secretion evoked by caffeine was small, being less than 10% of the response to strong agonists. Caffeine-induced tyrosine hydroxylase activation was not mimicked or prevented by phosphodiesterase inhibition with isobutylmethylxanthine, nor was it mimicked by an equimolar concentration of sucrose. However, the effect of caffeine was prevented by depleting intracellular Ca(2+) stores by thapsigargin pretreatment, and reduced substantially by removing extracellular Ca(2+), by blocking Ca(2+) channels with Co(2+) or Ni(2+), or by inhibiting store-operated channels with 2-aminoethyl diphenylborate. It was not affected by inhibiting Ca(2+) entry through voltage-operated Ca(2+)-channels or by tetrodotoxin. The effect of caffeine was mimicked by acute thapsigargin treatment or by depleting intracellular Ca(2+) stores in Ca(2+)-free buffer and then reintroducing extracellular Ca(2+). The results indicate that mobilizing store Ca(2+) with caffeine is a very effective mechanism for activating tyrosine hydroxylase and that the majority of this response depends on extracellular Ca(2+) entry through store-operated channels. They also suggest that extracellular Ca(2+) entry through such channels regulates cellular responses differently to Ca(2+) entry through voltage-operated Ca(2+) channels.