Stephanie W. Watts

Vascular Gap Junctional Communication Is Increased in Mineralocorticoid-Salt Hypertension

Cells rely on gap junctions for intercellular communication, which is important for growth and contractility. For example, gap junctional communication in the uterus increases near parturition, with a concomitant increase in oscillatory contractions. Because arterial responsiveness to contractile agonists is increased in hypertension, we tested the hypothesis that gap junctional communication is increased in hypertension. We examined thoracic aortas from deoxycorticosterone acetate (DOCA)-salt hypertensive and sham normotensive rats using isolated tissue baths and Western blotting techniques. The concentration of 5-hydroxytryptamine necessary to produce a threshold response was significantly lower in aortas from DOCA-salt (4 nmol/L) compared with sham (100 nmol/L) rats; this was also true for norepinephrine and KCl. In these same aortas, the appearance of spontaneous oscillatory contractions, which are sensitive to the gap junctional inhibitor heptanol (0.3 mmol/L), was more frequent in DOCA-salt arteries (93% versus 14% in sham). Heptanol (1 mmol/L) normalized the DOCA-salt aortic contraction to 5-hydroxytryptamine to levels similar to those of the response of the sham aorta in the presence of heptanol. Western analyses revealed that the density of connexin43 immunoreactivity, the connexin being a constituent of gap junctions, was found to be threefold more abundant in aortic homogenates of DOCA-salt rats compared with that of sham rats. This finding supports the hypothesis that gap junctional communication is increased in hypertension, at least at the protein level. We speculate that this increase results in a portion of the increased vascular reactivity and appearance of contractile oscillations in vascular smooth muscle.

Lead acetate-induced contraction in rabbit mesenteric artery: Interaction with calcium and protein kinase C

Animal studies have demonstrated that low-level lead exposure produces hypertension and that lead can cause contraction of vascular smooth muscle directly. The physiological effects of lead have been associated with both stimulation and inhibition of protein kinase C (PKC). Given that vascular smooth muscle contractility is generally enhanced when protein kinase C is activated, we have tested the hypothesis that lead contracts vascular smooth muscle via stimulation of PKC. Helically-cut strips of rabbit mesenteric artery were mounted in muscle baths for measurement of isometric force development. Cumulative addition of lead acetate (10(-10)-10(-3) M) to the muscle bath produced contractions (concentration necessary to produce half-maximal response -log EC50 = 5.16 +/- 0.07). Maximal contraction to lead acetate in arteries denuded of endothelium did not differ from those in intact vessels, supporting the hypothesis that lead-induced contraction is an endothelium-independent event. Contractions to lead acetate were potentiated by the PKC activators, phorbol 12-myristate 13-acetate (TPA; 3 x 10(-7) M) and mezerein (3 x 10(-7) M), as indicated by leftward shifts in the concentration-response curve and increase in the potency of lead (-log EC50 with TPA: 6.94 +/- 0.07; -log EC50 with mezerein: 6.07 +/- 0.04). H-7 (6 x 10(-6) M), an inhibitor of PKC, decreased maximal contraction to lead approximately 65% and slightly, but insignificantly, decreased the potency of lead (-log EC50 = 4.82 +/- 0.1). The inactive phorbol ester, phorbol 12-myristate 13-acetate 4-0-methyl ether (1 x 10(-6) M), did not alter contractile responses to lead (-log EC50 = 4.92 +/- 0.09). Vascular contraction to lead partially depends on extracellular calcium as the L-type voltage-gated calcium channel antagonist, verapamil (3 x 10(-6) M), decreased lead-induced contractions by 50%. These data indicate that lead interacts with PKC in an endothelium-independent, calcium-dependent manner to cause vascular smooth muscle contraction and suggest that lead-induced increases in vascular contractility may play a role in lead-induced hypertension.

The role of gap junctional communication in contractile oscillations in arteries from normotensive and hypertensive rats

Objectives: To test the hypotheses that vascular supersensitivity correlates with the appearance of contractile oscillations; vascular oscillations are mediated by gap junctions; and gap junctional communication is altered in the vasculature in stroke-prone spontaneously hypertensive (SHRSP) compared with Wistar-Kyoto (WKY) rats. Design and methods: Helical strips of mesenteric and tail arteries from SHRSP and WKY rats were mounted in tissue baths for measurement of isometric force. Cultures of mesenteric arterial cells were used for measurement of Lucifer yellow dye transfer and abundance of connexin43 mRNA, a monomer of gap junctions. Results: Mesenteric arteries from SHRSP that displayed spontaneous oscillations were more sensitive to the contractile agonist 5-hydroxytryptamine than those from SHRSP and WKY rats that displayed no oscillations. In addition, SHRSP tail arteries displayed norepinephrine-induced oscillations. The putative gap junction up-regulator tetraethylammonium (10~3-1 CH mol/l) induced oscillations (1-5 cycle/min) in arteries from both rat strains. These oscillations were not altered by endothelium removal or phentolamine and were blocked by heptanol (10~3 mol/l). Although tetraethylammonium and heptanol caused similar effects in both arteries, heptanol-sensitive agonist-induced oscillations persisted only in the tail artery of SHRSP Tetraethylammonium increased dye transfer between between contiguous cells approximately 35% above basal levels both for SHRSP and WKY cells. In both cell lines, heptanol reduced basal- and tetraethylammonium-stimulated dye transfer. Total RNA from WKY rat and SHRSP cultured cells hybridized strongly and to a similar magnitude with a complementary DNA probe for messenger RNA for connexin43. Conclusions: Gap junctional communication is important in vascular reactivity and might play a part in the development of oscillations. Altered gap junctional communication could not be demonstrated in cell cultures nor in some contractile experiments. It is possible that the culture conditions failed to mimic conditions in vivo that differentially regulate gap junctions in the hypertensive state, but it is also possible that gap junctional activity is not abnormal in hypertension.

5-Hydroxytryptamine2B Receptor Mediates Contraction in the Mesenteric Artery of Mineralocorticoid Hypertensive Rats

Vascular responsiveness to 5-hydroxytryptamine (5-HT) is dramatically increased in hypertension. The hypothesis that augmented vasoconstriction to 5-HT in hypertension is due to a change in receptor subtype on vascular myocytes was tested. Mesenteric arteries from deoxycorticosterone acetate (DOCA)-salt hypertensive (systolic blood pressure > 180 mm Hg) and sham normotensive (systolic blood pressure < 130 mm Hg) rats were mounted in isolated tissue baths for measurement of isometric contractile force. The receptor mediating contraction in isolated mesenteric arteries from sham and DOCA-salt hypertensive rats is a member of the 5-HT2 family based on rank order of agonist potency (5-HT = alpha-methyl-5-HT [5-HT2 receptor agonist]>tryptamine>5-hydroxykynuramine). 5-HT was approximately 10-fold more potent in contracting mesenteric arteries from DOCA-salt hypertensive rats compared with arteries from sham normotensive rats. The tryptophan metabolite kynuramine, which possesses significant contractile activity at the 5-HT2B receptor, contracted hypertensive arteries significantly (50% of 5-HT maximum) but not sham arteries. Ketanserin (5-HT2A antagonist) competitively inhibited contraction to 5-HT in arteries from normotensive rats (-log dissociation constant [mol/L]; pKB = 8.54) but not from hypertensive rats (pKB > 6.5). Moreover, contraction to kynuramine was not blocked by ketanserin. Thus, under normal conditions, 5-HT2A receptors mediate contraction to 5-HT. However, in DOCA-salt hypertension, ketanserin-insensitive 5-HT2 receptors, possibly 5-HT2B receptors, mediate mesenteric arterial contraction to 5-HT.

Gap junctional communication and vascular smooth muscle reactivity: Use of tetraethylammonium chloride

Oscillatory contractions in uterine smooth muscle are mechanistically related to gap junction complex formation. We have tested the hypothesis that agonist-induced oscillations in vascular smooth muscle are also mediated by gap junctions and that gap junctions are important for vascular smooth muscle cell communication. Total RNA from cultured Wistar-Kyoto rat (WKY) mesenteric arterial cells hybridized strongly with a cDNA probe for the message for connexin43, a monomer of the gap junction. In these same cells, the quaternary ion tetraethylammonium (TEA) (10 mM) increased Lucifer yellow dye transfer between contiguous cells, a measure of cell-to-cell communication via gap junctions, approximately 35% above basal levels. Heptanol, an established inhibitor of gap junction communication, completely blocked both basal- and TEA-stimulated dye transfer between neighboring cells. In other experiments, helical strips of superior mesenteric and tail arteries from WKY rats were mounted in tissue baths for measurement of isometric contractile force. TEA (10(-3)-10(-1) M) induced oscillatory contractions (1-5 cycle/min) in both mesenteric and tail arteries. Removal of endothelium did not affect the pattern of TEA-stimulated oscillations. Oscillations to TEA were blocked in a concentration-dependent manner in both arteries by heptanol (10(-7)-10(-3) M). Heptanol (10(-3) M) also significantly reduced (40%) acetylcholine-induced relaxation in the mesenteric artery (contracted with phenylephrine).(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of Ramipril on Contractile Oscillations in Arteries from Genetically Hypertensive Rats

We have tested the hypothesis that altered vascular reactivity, specifically the appearance of spontaneous and BayK 8644 (L-type voltage gated calcium channel agonist)-induced oscillations in the carotid artery and the sarcoplasmic reticulum Ca(2+)-ATPase inhibitor cyclopiazonic acid (CPA)-induced oscillations in the aorta from stroke-prone spontaneously hypertensive rats (SHRS), are dependent upon angiotensin II production early in life. SHRSP and normotensive Wistar-Kyoto (WKY) rats were treated from 6-10 weeks of age with vehicle, hydralazine/hydrochlorothiazide (used as a control for lowered blood pressure) or the angiotensin converting enzyme inhibitor ramipril (3 mg/kg/day). Systolic blood pressures were measured weekly in rats from 6 to 17 weeks of age. In SHRSP (at 17 weeks of age), ramipril-treatment but not hydralazine/hydrochlorothiazide attenuated the long term expression of elevated systolic blood pressure in adult SHRSP while blood pressures of all adult WKY rats were unaffected by any treatment. At 17 weeks, rats were killed and arteries removed for in vitro measurement of isometric contractile activity. Only the incidence of spontaneous oscillations (carotid artery) was affected by ramipril treatment; ramipril did not change the frequency of BayK 8644-induced oscillations in the artery or the frequency of CPA-induced oscillations in aorta from either SHRSP or WKY. These data indicate that while spontaneous oscillations in the carotid artery may be dependent on an angiotensin II-sensitive mechanism during development, agonist-induced oscillations (CPA and BayK 8644) appear not to be angiotensin II-dependent. Thus, not all of the contractile oscillations which appear in vascular smooth muscle from SHRSP are angiotensin II-dependent, suggesting that some of these vascular abnormalities may develop at a time separate from that in which increased blood pressure is firmly established and may not be associated with the for maintenance of elevated blood pressure.

Enhanced Vascular Responsiveness to Bay K 8644 in Mineralocorticoid- and N-Nitro Arginine-Induced Hypertension

The present study evaluates the response to the L-type voltage gated calcium channel agonist Bay K 8644 in two forms of experimental hypertension (mineralocorticoid- and hypertension induced by the nitric oxide synthase inhibitor N omega-nitro-L-arginine (N-Nitro arginine)) and under conditions of acute stretch. These studies test the hypothesis that increased L-type calcium channel activity in vasculature is a hallmark or general characteristic of hypertension. Male Sprague-Dawley rats were made hypertensive by subcutaneous implantation of deoxycorticosterone acetate (200 mg/kg DOCA) and given normal or high salt water (1% NaCl + 0.2% KCl); other rats were made hypertensive by ingestion of N-Nitro arginine (2% in water). Systolic blood pressures (SBP) were taken by the standard tail cuff method. Following development of hypertension, rats were anesthetized, and aortae or mesenteric arteries were isolated for measurement of isometric contractile force. Cumulative concentration response curves to Bay K 8644 (10(-10) to 10(-6) M), KCl (6 to 100 mM), or phenylephrine (10(-10)-3 x 10(-7) M) were evaluated. Isolated mesenteric arteries from rats given both DOCA and salt were most sensitive to Bay K 8644 (SBP = 191 +/- 6 mmHg, -log EC50 = 7.78 +/- 0.13), followed by rats receiving high salt alone (SBP = 118 +/- 6 mmHg, -log EC50 = 7.30 +/- 0.17), DOCA alone (SBP = 152 +/- 2 mmHg, -log EC50 = 7.25 +/- 0.15), and finally normal sham rats (SBP = 111 +/- 5 mm Hg, -log EC50 > or = 6.80 +/- 0.10). These data indicate that both DOCA and salt intake can independently influence responsiveness to Bay K 8644.(ABSTRACT TRUNCATED AT 250 WORDS)

Photorelaxation Is Not Attenuated by Inhibition of the Nitric Oxide-cGMP Pathway

Photorelaxation of arteries by ultraviolet (UV) light is hypothesized to result from nitric oxide (NO) released from photoactivable stores. Recently, a study reported enhanced photorelaxation of aortic tissue from rats administered the NO synthase (NOS) inhibitor N omega-nitro-L-arginine (L-NNA). Presumably, the potentiated photorelaxation was due to NO generated from UV-light-induced decomposition of the NO2 moiety of L-NNA. However, we hypothesized that photorelaxation is: (1) not the result of NO synthesis and subsequent activation of guanylate cyclase and (2) not due to hyperpolarization induced by NO or any other factor. Endothelium-denuded rat aortic rings were suspended in isolated baths for isometric force measurement. Rings were exposed to UV light (366 nm) before addition of phenylephrine or KCI, and then at each agonist concentration during a cumulative concentration response curve. NOS inhibition by L-NNA and L-thiocitrulline, which lacks an NO2 group, enhanced photorelaxation of basal myogenic tone and contraction to phenylephrine (EC70). Furthermore, relaxation of a maximum phenylephrine-induced contraction to the NO donor S-nitroso-N-acetyl-D,L-penicillamine during UV light exposure was not altered by incubation of rings with L-NNA or tissues from animals fed L-NNA. These data demonstrate that NO is not produced endogenously or from the breakdown of L-NNA to result in photo-relaxation. Methylene blue (MB) did not alter photorelaxation, suggesting that cGMP is not essential to the response. MB and L-NNA together potentiated photorelaxation of basal myogenic tone and phenylephrine-induced contraction. Photorelaxation of KCl-induced contraction was unaltered, indicating that hyperpolarization does not contribute to the relaxation. Photorelaxation of basal myogenic tone and KCl-induced contraction excludes the possibility that UV light is interfering with agonist-receptor binding. Collectively, these results refute the hypotheses that photorelaxation results from activation of the NO-cGMP pathway, release of a hyperpolarization factor, or inhibition of drug-receptor interaction. Interestingly, photorelaxation may be inhibited by NO-cGMP pathway activation, uncovering a novel effect of this messenger system on vascular reactivity.

Effect of ramipril on α-adrenoceptor-mediated oscillatory contractions in tail artery of hypertensive rats

Recent studies indicate that norepinephrine-induced contractile oscillations in the tail artery from stroke-prone spontaneously hypertensive rats (SHRSP) may be a vascular phenomenon independent of blood pressure level. The objectives of this study were: (1) to characterize pharmacologically the alpha-adrenoceptor mediating norepinephrine-induced oscillations in tail artery; and (2) to investigate the relationship between blood pressure level, altered by treatments with hydralazine/hydrochlorothiazide or the angiotensin converting enzyme inhibitor ramipril, and the observation of norepinephrine-induced oscillations in tail artery. The alpha 2-adrenoceptor agonists clonidine and guanabenz potently stimulated oscillatory contractions in the tail artery while the alpha 1-adrenoceptor agonists phenylephrine and methoxamine were considerably less potent. Yohimbine, an alpha 2-adrenoceptor antagonist, but not the alpha 1-adrenoceptor antagonist prazosin demonstrated high affinity for the receptor mediating norepinephrine-induced oscillatory contractions. These results support the hypothesis that norepinephrine-induced oscillatory contractions in the tail artery from SHRSP occur primarily through stimulation of alpha 2-adrenoceptors. Ramipril lowered blood pressure in SHRSP after 4 weeks of treatment during 6-10 weeks of life but did not alter the ability of the alpha 2-adrenoceptor agonist clonidine (10(-5) M) to induce contractile oscillations in tail arteries from SHRSP, indicating these oscillations are not a secondary effect of high blood pressure. These studies suggest that norepinephrine-induced oscillations in tail artery from SHRSP may be a vascular trait separate and distinct from blood pressure level and angiotensin II expression early in life.

Non-cGMP, Paracrine Effects of Nitric Oxide in the Vasculature

Nitric oxide (NO) is as an important endogenous modulator of blood vessel function (1,2). NO released from vascular endothelial cells acts in a paracrine fashion on adjacent vascular smooth muscle cells (VSMC) to activate soluble guanylyl cyclase and generate cyclic guanosine 3’,5’ monophosphate (cGMP) (2). cGMP, in turn, mediates many of NO-mediated actions in VSMC. However, a number of cGMP-independent effects have been observed following NO stimulation, indicating that activation of guanylyl cyclase accounts for only a portion of the effects of NO.