[Identification of a Novel Calcium (Ca

Calcium (Ca^(2+))-activated chloride channel accessories (CLCAs) are putative anion channel-related proteins with diverse physiological functions. Exploring CLCA diversity is important for prediction of gene structure and function. In an effort to identify novel CLCA genes in Xenopus laevis, we successfully cloned and characterized a Xenopus laevis cDNA predicted to encode the xCLCA3 gene. Cloning of xCLCA3 was achieved by computational analysis, rapid amplification of cDNA ends (RACE), and a tissue distribution analysis by semi-quantitative reverse transcription (RT) PCR or real-time PCR. We obtained a 2958 bp xCLCA3 cDNA sequence with an open reading frame encoding 943 amino acids. According to the primary structure analysis, xCLCA3 contains a predicted signal sequence, multiple sites of N-linked (N-) glycosylation, N-myristoylation, PKA, PKC, and casein kinase II phosphorylation sites, five putative hydrophobic segments, and the HExxH metalloprotease motif. Additionally, the transmembrane prediction server yielded a preserved N-terminal CLCA domain and a von Willebrand factor type A domain with one transmembrane domain in the C-terminal region. Expression analysis showed that xCLCA3 is expressed in a number of tissues, with strong expression in the brain, colon, small intestine, lung, kidney, and spleen, and poor expression in the heart and liver. These results suggest that xCLCA3 may be a candidate CLCA family member as well as a metalloprotease, rather than just an ion channel accessory protein.

Modulation of thiopental-induced vascular relaxation and contraction by perivascular adipose tissue and endothelium.

BACKGROUND: Thiopental induces relaxation of vascular smooth muscle cells through its direct and/or indirect vasodilator effects. The perivascular adipose tissue (PVAT) and the endothelium are known to attenuate vascular contraction, and we have recently reported that PVAT potentiates the relaxation effect of propofol through endothelium-dependent and -independent mechanisms. Here, we studied the mechanisms of thiopental-induced vascular responses in relation to the involvement of PVAT and endothelium. METHODS: Thoracic aortic rings from male Wistar rats were prepared with or without PVAT (PVAT+ and PVAT-) and with an intact endothelium (E+) or with the endothelium removed (E-) for functional studies. The contraction and relaxation responses of these vessels to thiopental in the presence of agonists and various receptor antagonists and channel blockers were studied. RESULTS: In vessels pre-contracted with phenylephrine or KCl, thiopental-induced relaxation was highest in vessels denuded of both PVAT and the endothelium. PVAT attenuated the relaxation response to thiopental, and this attenuation effect was reduced by both angiotensin II (Ang II) type 1 receptor antagonists CV-11974 (2-n-butyl-4-choloro-5-hydroxymethyl-1-[2'-(1H-tetrazol-5-yl)biphenyl-methyl]-imidazole) or losartan and the angiotensin-converting enzyme inhibitor enalaprilat. Thiopental at high concentration (3 × 10(-3) M) caused a contraction through an endothelin-dependent mechanism. CONCLUSIONS: Thiopental induced relaxation in rat aorta through an endothelium-independent pathway and the presence of PVAT, endothelium, or both attenuated this relaxation response through Ang II-dependent and endothelin-dependent mechanisms, respectively.

Induction of melanoma cell apoptosis and inhibition of tumor growth using a cell-permeable Survivin antagonist.

The inhibitor of apoptosis gene family member Survivin is highly expressed in most tumors, and appears to be a promising target for cancer therapy. Although a variety of Survivin antagonists have been shown to induce apoptosis in malignant cells, the potential utility of these agents is limited by inefficient delivery and cell impermeability. We generated recombinant fusion proteins containing the TAT protein transduction domain and either wild-type Survivin (TAT-Surv-WT) or a dominant-negative mutant (TAT-Surv-T34A). The TAT-Surv proteins were purified by sequential affinity and ion-exchange chromatography, and at 30 nM concentration demonstrated rapid entry into cells at 30 min. Whereas TAT-Surv-WT had minimal effect on YUSAC2 or WM793 melanoma cells, TAT-Surv-T34A induced cell detachment, DNA fragmentation, caspase-3 activation and mitochondrial release of apoptosis-inducing factor at low microM concentrations. Intraperitoneal (i.p.) injection of mice bearing subcutaneous YUSAC2 xenografts with TAT-Surv-T34A (10 mg/kg) was associated with rapid tumor accumulation at 1 h, and increased tumor cell apoptosis and aberrant nuclei formation in situ. Repeated i.p. injection of TAT-Surv-T34A resulted in a 40-50% reduction in growth and mass of established tumors, compared to those similarly injected with saline buffer or TAT-Surv-WT. These studies demonstrate the feasibility of systemic tumor treatment using a cell-permeable Survivin antagonist.

Modulation of vascular function by perivascular adipose tissue: the role of endothelium and hydrogen peroxide.

BACKGROUND AND PURPOSE: Perivascular adipose tissue (PVAT) attenuates vascular contraction, but the mechanisms remain largely unknown. The possible involvement of endothelium (E) and hydrogen peroxide (H2O2) was investigated. EXPERIMENTAL APPROACH: Aortic rings from Wistar rats were prepared with both PVAT and E intact (PVAT+ E+), with either PVAT or E removed (PVAT- E+, or PVAT+ E-), or with both removed (PVAT- E-) for functional studies. Nitric oxide (NO) production was measured. KEY RESULTS: Contraction to phenylephrine and 5-HT respectively was highest in PVAT- E-, lowest in PVAT+ E+, and intermediate in PVAT+ E- or PVAT- E+. In bioassay experiments, transferring bathing solution incubated with a PVAT+ ring (donor) to a PVAT- ring (recipient) induced relaxation in the recipient. This relaxation was abolished by E removal, NO synthase inhibition, scavenging of NO, high extracellular K+, or blockade of calcium-dependent K+ channels (K(Ca)). The solution stimulated NO production in isolated endothelial cells and in PVAT- E+ rings. In E- rings, the contraction to phenylephrine of PVAT+ rings but not PVAT- rings was enhanced by catalase or soluble guanylyl cyclase (sGC) inhibitor, but reduced by superoxide dismutase and tiron. In PVAT- E- rings, H2O2 attenuated phenylephrine-induced contraction. This effect was counteracted by sGC inhibition. NO donor and H2O2 exhibited additive inhibition of the contraction to phenylephrine in PVAT- E- rings. CONCLUSION: PVAT exerts its anti-contractile effects through two distinct mechanisms: (1) by releasing a transferable relaxing factor which induces endothelium-dependent relaxation through NO release and subsequent K(Ca) channel activation, and (2) by an endothelium-independent mechanism involving H2O2 and subsequent activation of sGC.

Collective cell migration over long time scales reveals distinct phenotypes.

INTRODUCTION: Migratory phenotypes of metastasizing tumor cells include single and collective cell migration. While migration of tumor cells is generally less cooperative than that of normal epithelial cells, our understanding of precisely how they differ in long time behavior is incomplete. OBJECTIVES: We measure in a model system how cancer progression affects collective migration on long time scales, and determine how perturbation of cell-cell adhesions, specifically reduced E-cadherin expression, affects the collective migration phenotype. METHODS: Time lapse imaging of cellular sheets and particle image velocimetry (PIV) are used to quantitatively study the dynamics of cell motion over ten hours. Long time dynamics are measured via finite time Lyapunov exponents (FTLE) and changes in FTLE with time. RESULTS: We find that non-malignant MCF10A cells are distinguished from malignant MCF10CA1a cells by both their short time (minutes) and long time (hours) dynamics. In addition, short time dynamics distinguish non-malignant E-cadherin knockdown cells from the control, but long time dynamics and increasing spatial correlations remain unchanged. DISCUSSION: Epithelial sheet collective behavior includes long time dynamics that cannot be captured by metrics that assess cooperativity based on short time dynamics, such as instantaneous speed or directionality. The use of metrics incorporating migration data over hours instead of minutes allows us to more precisely describe how E-cadherin, a clinically relevant adhesion molecule, affects collective migration. We predict that the long time scale metrics described here will be more robust and predictive of malignant behavior than analysis of instantaneous velocity fields alone.

Subcellular fractionation of pig coronary artery smooth muscle.

A detailed procedure for subcellular fractionation of the smooth muscle from pig coronary arteries based on dissection of the proper tissue, homogenization, differential centrifugation and sucrose density gradient centrifugation is described. A number of marker enzymes and Ca2+ uptake in presence or absence of oxalate, ruthenium red and azide were studied. The ATP-dependent oxalate-independent azide- or ruthenium red-insensitive Ca2+ uptake, and the plasma membrane markers K+-activated ouabain-sensitive p-nitrophenylphosphatase, 5'-nucleotidase and Mg2+-ATPase showed maximum enrichment in the F2 fraction (15-28% sucrose) which was also contaminated with the endoplasmic reticulum marker NADPH: cytochrome c reductase, and to a small extent with the inner mitochondrial marker cytochrome c reductase, and also showed a small degree of oxalate stimulation of the Ca2+ uptake. F3 fraction (28-40% sucrose) was maximally enriched in the ATP- and oxalate-dependent azide-insensitive Ca2+ uptake and the endoplasmic reticulum marker NADPH: cytochrome c reductase but was heavily contaminated with the plasma membrane and the inner mitochondrial markers. The mitochondrial fraction was enriched in cytochrome c oxidase and azide- or ruthenium red-sensitive ATP-dependent Ca2+ uptake but was heavily contaminated with other membranes. Electron microscopy showed that F2 contained predominantly smooth surface vesicles and F3 contained smooth surface vesicles, rough endoplasmic reticulum and mitochondria. The ATP-dependent azide-insensitive oxalate-independent and oxalate-stimulated Ca2+ uptake comigrated with the plasma membrane and the endoplasmic reticulum markers, respectively, and were preferentially inhibited by digitonin and phosphatidylserine, respectively. This study establishes a basis for studies on receptor distribution and further Ca2+ uptake studies to understand the physiology of coronary artery vasodilation.

Alterations of elastin and elastase-like activities in aortae of diabetic rats.

The elastin content of the aortic muscle and the elastase-like activity of the extracts of aortic muscle were studied in spontaneously diabetic BB rats and in rats made diabetic by a single bolus i.v. injection of alloxan. In both modes of diabetes, the total alkaline-insoluble aortic elastin content was significantly reduced in diabetic rats compared to that in the corresponding control rats. This reduction in aortic elastin was also accompanied by a consistent increase in the elastase-like activities of the aortic extracts prepared from the same tissues. Such a reciprocal relationship between aortic elastin content and elastase-like activity has previously been reported in rats with malignant hypertension. Since the rats used in this study were not hypertensive, the altered elastin metabolism observed in this work is likely to be a manifestation of diabetic disease and may in part account for the vascular changes associated with diabetes mellitus.

Morphology of cerebral arteries.

A comparison of the major cerebral arteries between humans and rats shows many similarities, including anomalies in their general organization, the structure of these vessels at the light and electron microscope levels and their morphological changes associated with cerebral vascular diseases. The general organization of the major cerebral arteries shows the following main differences between humans and rats. In rats, the internal carotid arteries have become an integral part of the circle of Willis. In the anterior cerebral arteries, a common variation in humans is the underdevelopment of one of the two arteries, whereas in rats, buttonhole-like structures are common in one or both arteries. The anterior communicating artery present in humans is absent in rats. The olfactory artery is prominent in rats, but absent in humans. The posterior communicating artery in humans is the most variable component of the circle of Willis, being asymmetric in its origin, diameters and branches. Similarly, the posterior cerebral arteries in rats often exhibit asymmetrical origin from the basilar artery. There was some confusion in the literature regarding the name of the posterior cerebral arteries in rats, but this was caused mainly by misquotations and incorrect interpretations of the papers. In humans, most aneurysms occur in the anterior half of the circle of Willis, and the incidence is higher in females than males; the middle cerebral artery is most often the one to become occluded, and the vertebral arteries are common sites for thrombosis. The various channels that constitute collateral circulation in humans provide a margin of safety, so that in case of cerebral occlusion due to thrombosis, atherosclerosis, or vasospasm related to hemorrhage, blood supply to the affected area can be maintained through these collaterals. Collateral circulation is also present in rats. However, in rats, information on the presence of various types of aneurysms, their location and frequency in normal and experimental models of hypertension and stroke is still lacking. Cerebral arteries from humans and rats are characterized by the absence of external elastic lamina, as compared with systemic arteries. A type of multipolar cell resembling the interstitial cell of Cajal is present in the cerebral arteries of humans. Its function is unknown. Earlier reports of cerebral valves have been shown to represent intimal cushions near the branching points of the cerebral arteries. Intravascular bridges present in human cerebral arteries, have not been reported in rats. Finally, the presence of vascular remodeling, as found in the cerebral arterioles of hypertensive rats, remains to be established in the cerebral arterioles of human hypertensives.(ABSTRACT TRUNCATED AT 400 WORDS)

Effect of hydralazine on the mesenteric vasculature of hypertensive rats.

To test whether structural alterations observed in the mesenteric vasculature of Wistar-Kyoto spontaneously hypertensive rats (SHR) were dependent on the presence of hypertension, male SHR and Wistar-Kyoto normotensive (WKY) rats were treated in utero and postnatally with hydralazine up to 28 weeks of age. Treated SHR, WKY, and untreated WKY rats had comparable blood pressures that were less than those of untreated SHR. Treatment altered the dimensions of the superior mesenteric, intermediate-sized, and small arteries of the mesenteric vasculature. In the case of the superior mesenteric artery and intermediate vessels, hydralazine treatment increased the lumen and medial cross-sectional areas of the arteries in WKY rats and slightly decreased both parameters in SHR. Within the small arteries, treatment significantly increased the lumen size in SHR but not WKY rats and had no significant effect on the media of the vessels. Despite the above alterations, the media-to-lumen cross-sectional area ratios remained significantly elevated in SHR over WKY rats in both the treated and control groups of animals within all classes of arteries. The results indicate that there is an inherent increase in the quantity of media surrounding the arteries of SHR when compared with WKY rats that cannot be abolished by normalizing the blood pressure in utero and postnatally with hydralazine treatment. In SHR, such changes persist not only in arteries that exhibit an increase in the media-to-lumen ratio before hypertension but also in the superior mesenteric artery in which an increase in the ratio occurs after hypertension development.

Structural and functional analysis of small arteries from young spontaneously hypertensive rats.

We studied structural and functional changes of small muscular arteries from the mesenteric vascular bed of young spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto rats (WKY) using a new morphometric protocol involving the use of confocal microscopy and a pressurized artery system. At 3 and 4 weeks of age, systolic pressure of SHR and WKY was similar; however, significant structural changes in the mesenteric vasculature were already present in SHR. Arteries fixed under pressure in vitro from SHR had a larger medial volume and increased number of smooth muscle cell layers but similar lumen size compared with arteries from WKY in maximally relaxed conditions. Functional studies showed that SHR arteries contracted more in response to stimulation by KCl and norepinephrine, resulting in a significantly smaller lumen size in these vessels than in those from WKY. SHR arteries precontracted with KCl were also able to maintain a smaller lumen diameter than WKY arteries when challenged with increasing pressure levels. No difference in the sensitivity of response of these arteries to norepinephrine stimulation was found. At 3 and 4 weeks of age, mesenteric arteries from some SHR and WKY were not responsive to periarterial nerve stimulation, and the number of responders was higher in the WKY than SHR. However, a greater degree of contraction was found in SHR arteries responding to field stimulation at 4 weeks than in WKY arteries. We conclude that there is a temporal difference in the rate of functional maturation of the innervation in SHR arteries compared with WKY arteries. Structural changes of the small muscular arteries, caused by an increase in the medial volume, and increased number of smooth muscle cell layers are primary changes that contribute to the development of hypertension in the SHR because these changes are present at the age when blood pressure is similar in SHR and WKY.

Peptide-containing nerves around blood vessels of stroke-prone spontaneously hypertensive rats.

The distribution and density of nerves containing vasoactive intestinal polypeptide, substance P, and neuropeptide Y around the cerebral and peripheral blood vessels of stroke-prone spontaneously hypertensive rats (SHRSP) and normotensive Wistar-Kyoto rats (WKY) were studied using an indirect immunofluorescence technique. Neonatal sympathectomy of SHRSP with anti-nerve growth factor and guanethidine was also carried out to study the effect of sympathectomy on the distribution of these nerves. Vasoactive intestinal polypeptide nerve density was higher in the veins and superior mesenteric artery of SHRSP than of WKY and lower in the cerebral arteries of SHRSP than of WKY, but no difference was found in the muscular mesenteric arteries. Sympathectomy reduced the density of these nerves in all the peripheral vessels but had little effect on the cerebral arteries. Density of substance P nerves was similar between SHRSP and WKY in the peripheral vessels but higher in the cerebral arteries of WKY than of SHRSP. Sympathectomy reduced the density of these nerves in the peripheral vessels but increased the density in some cerebral arteries of SHRSP. Neuropeptide Y nerve density was higher in the peripheral blood vessels of SHRSP than of WKY, and no difference was found in the cerebral arteries. Sympathectomy almost completely removed these nerves in the peripheral vessels but had no effect on the cerebral arteries. We suggest that some of the differences in nerve density between SHRSP and WKY, especially those in the peripheral blood vessels, may be related to the development of hypertension in the SHRSP.

Antihypertensive effect of gamma-linolenic acid in spontaneously hypertensive rats.

The effects of chronic treatments of adult (aged 16-17 weeks) spontaneously hypertensive rats (SHRs) with different doses of gamma-linolenic acid (GLA) on blood pressure, heart rate, and body weight were studied. Twice-daily injection of SHRs with GLA lowered systolic blood pressure from 175 +/- 4 to 145 +/- 4 mm Hg within 1 week; systolic blood pressure in all three treated groups became stabilized in the normotensive range after 2 weeks of treatment. Control SHRs injected with olive oil showed only a transient decrease in systolic blood pressure on the third day. Heart rate and body weight were not affected by GLA treatment. Withdrawal of GLA treatment resulted in a rapid rise in systolic blood pressure within 1 day from 140 +/- 3 to 165 +/- 3 mm Hg, and it stabilized after 1 week at 191 +/- 5 mm Hg in the three experimental groups. A rapid increase in systolic blood pressure from 175 +/- 5 to 203 +/- 5 mm Hg was also observed in the control group treated with olive oil 1 day after the withdrawal of the treatment. Addition of aspirin (3 mg/kg) with the GLA treatment in olive oil abolished the antihypertensive effect of GLA. In contrast, once-daily treatment with GLA also lowered systolic blood pressure of the SHR, but blood pressure was still in the hypertensive range (170 +/- 6 mm Hg). Systolic blood pressure of control SHRs treated with olive oil was not affected. Plasma from untreated SHRs contained a small amount of GLA. One hour after the injection, the plasma level of GLA increased. We conclude that GLA when given twice daily is an effective antihypertensive agent in the SHR.

AT1 receptor antagonist treatment caused persistent arterial functional changes in young spontaneously hypertensive rats.

The effects of chronic treatment with an AT1 receptor antagonist (L-158,809) on hypertension development and cardiovascular changes were studied in spontaneously hypertensive rats (SHR) and Wistar-Kyoto rats (WKY). L-158,809 treatment (0.6 mg/kg PO) was initiated at 3 weeks of age and lasted 12 weeks, to 15 weeks of age. The treatment prevented hypertension development in the SHR (systolic blood pressure, BP, of 136+/-1 mm Hg compared with 198+/-3 mm Hg in control SHR), and lowered the BP of WKY (99+/-2 vs 128+/-1 mm Hg in control WKY). Treatment significantly reduced the heart weight in SHR and WKY. Ten weeks after treatment withdrawal (25 weeks of age), BP had increased in SHR and WKY to 172+/-8 and 117+/-3 mm Hg, respectively. Body weight and kidney weight were not affected by the treatment. Mesenteric arteries from treated SHR were less responsive than control SHR arteries to periarterial nerve stimulations at transmural pressures higher than 80 mm Hg (15 and 25 weeks). Control WKY arteries were less responsive than control SHR arteries at almost all transmural pressures tested (15 weeks) and to pressures greater than 80 mm Hg (25 weeks). Pretreatment of arteries with 10(-8) mol/L angiotensin II enhanced their response to nerve stimulation in vessels from control SHR and WKY (25 weeks) but not from treatment-withdrawn SHR and WKY. Treatment did not alter arterial reactivity in response to norepinephrine. Alteration in arterial structure due to L-158,809 treatment was found only when measured at a transmural pressure of 100 mm Hg. In conclusion, L-158,809 was effective in preventing hypertension during the treatment period, in reducing hypertension severity during the withdrawal period, and in persistently decreasing the reactivity of the arteries.

Structural and functional consequence of neonatal sympathectomy on the blood vessels of spontaneously hypertensive rats.

Neonatal sympathectomy of spontaneously hypertensive rats (SHR) and control Wistar-Kyoto rats (WKY) was performed by a combined treatment with antiserum to nerve growth factor and guanethidine during the first 4 weeks after birth. The development of hypertension was completely prevented in the treated SHR: at 28 to 30 weeks of age, systolic blood pressure of treated SHR was 139 +/- 2 mm Hg as compared with 195 +/- 8 mm Hg in untreated SHR. The extent of sympathectomy was verified by histofluorescence. Fluorescence histochemistry for catecholamine-containing nerves showed a complete absence of adrenergic nerves in the mesenteric arteries of treated rats. A supersensitivity to norepinephrine was exhibited by mesenteric arteries, anococcygeus muscle, and tail arteries from the treated SHR and WKY. In the mesenteric vascular bed, maximal response to norepinephrine was significantly reduced by sympathectomy. Sympathectomy also abolished the responses (e.g., generation of excitatory junctional potentials) of tail arteries to electrical stimulation of perivascular nerves. Morphometric measurements of three categories of mesenteric arteries showed that sympathectomy had no effect on the hypertrophic change of smooth muscle cells in the conducting vessels, but it prevented the hyperplastic changes of the muscle cells from reactive, muscular arteries and small resistance vessels. These results suggest that one of the primary roles of the overactive sympathetic nervous system in the development of hypertension in SHR is manifested through its trophic effect on the arteries of SHR. This trophic effect appears to cause a hyperplastic change in the smooth muscle cells in the reactive and resistance vessels, thereby contributing to the development of hypertension in older SHR.

Prevention of hypertension and vascular changes by captopril treatment.

Treatment of female spontaneously hypertensive rats (SHR) and control Wistar-Kyoto (WKY) rats with captopril was carried out by the addition of the drug in the drinking water throughout pregnancy and lactation and after weaning. At 28 weeks of age, average systolic blood pressure of treated SHR was 113 +/- 3 mm Hg, which was below that of control SHR (188 +/- 3 mm Hg) and WKY rats (124 +/- 3 mm Hg). Body weight and heart rate of the SHR were not affected by the treatment. Tissue level of catecholamines was increased by captopril treatment in the superior cervical ganglia but remained unchanged in the plasma, heart, mesenteric arteries, and the adrenal glands of both SHR and WKY rats. Left ventricular weight, wall thickness, and internal diameter of the left ventricle in the SHR were reduced by the treatment. Morphometric measurements of the mesenteric arteries showed that vascular alterations present in the control SHR were prevented by the treatment. In the superior mesenteric artery and large mesenteric artery, smaller lumen size at maximal relaxation found in the control SHR was normalized to the level of the WKY rats. Hypertrophy of the medial wall in the superior mesenteric, large and small mesenteric arteries, and an increase in the number of smooth muscle cell layers in the large mesenteric artery of the SHR were prevented by the treatment. Perfusion study of the mesenteric vascular bed showed that reactivity of these vessels to norepinephrine was reduced, and sensitivity to norepinephrine (as determined by the effective dose that causes 50% of maximal response) was increased in the SHR by captopril treatment. Sensitivity of the tail artery in response to norepinephrine was not altered by the treatment. We conclude that long-term treatment with captopril of SHR before and after birth prevented the development of hypertension, structural and functional alterations of the mesenteric arteries, and cardiac hypertrophy.

Effects of sarcoplasmic reticulum calcium pump inhibitors on vascular smooth muscle.

A dysfunctioning of Ca2+ pump ATPase in the sarcoplasmic reticulum in vascular smooth muscle has been proposed as a contributing factor for the development of genetic hypertension. In this study, we determined whether in vitro inhibition of the sarcoplasmic reticulum Ca2+ pump in vascular smooth muscle tissues and cultured cells isolated from aortas of spontaneously hypertensive rats (SHR) and Wistar-Kyoto (WKY) rats would elicit the known alterations of contractile function and cell growth. We found the following common vascular effects of thapsigargin and cyclopiazonic acid, which are known to be selective inhibitors of sarcoplasmic reticulum Ca(2+)-ATPase in a number of tissues including smooth muscle: (1) Both sarcoplasmic reticulum Ca2+ pump inhibitors diminished agonist-induced transient contraction in Ca(2+)-free medium (ie, contraction due to intracellular release of Ca2+) and enhanced nifedipine-sensitive contraction on readmission of Ca2+ (ie, Ca2+ influx via L-type channels); and (2) thapsigargin and cyclopiazonic acid inhibited the attachment of cultured aortic muscle cells to the substrate in a similar degree in both SHR and WKY cells, but SHR cells were more sensitive than WKY cells to the inhibition of cell proliferation by these two agents. The first effect may provide an explanation for several contractile abnormalities known to be associated with elevated cytosolic Ca2+ concentration, whereas the second effect suggests that elevation of cytosolic Ca2+ in aortic smooth muscle cells is not necessarily associated with or sufficient to account for the accelerated cellular proliferation in SHR. These results, however, further stress the functional importance of impairment of Ca2+ regulation in vascular smooth muscle cells in genetic hypertension.

Morphometric study of cerebral arteries from spontaneously hypertensive and stroke-prone spontaneously hypertensive rats.

OBJECTIVE: The importance of sympathetic innervation for the development of structural changes in the cerebral arteries of hypertensive animals was studied. DESIGN: Sympathetic denervation was induced with combined treatment from birth of antibody against nerve growth factor and guanethidine. Previous studies from our laboratory showed that this procedure not only caused a permanent denervation of the mesenteric arteries, but also prevented the development of hypertension in spontaneously hypertensive rats (SHR). METHODS: Morphometric measurement of the structural changes was carried out in the basilar, superior cerebellar, posterior cerebral and middle cerebral arteries from 28-week-old SHR, stroke-prone SHR, and normotensive Wistar-Kyoto rats. The results were compared with those obtained from cerebral arteries of sympathectomized rats. RESULTS: Total vascular wall cross-sectional area was significantly larger in the basilar and superior cerebellar arteries from hypertensive rats compared with normotensives. The change was characterized by an increase in the number of smooth muscle cell layers. There were also differences between the two hypertensive groups in some arteries. Sympathetic denervation attenuated the development of hypertension and vascular changes in some arteries. There was a positive linear correlation between blood pressure and medial cross-sectional area, and between blood pressure and the number of smooth muscle cell layers for the four arteries analysed. CONCLUSION: Sympathetic nerves have a trophic influence upon the remodelling of some cerebral arteries during the development of genetic hypertension.

Apoptosis in the muscular arteries from young spontaneously hypertensive rats.

OBJECTIVE: The purpose of this study was to test the hypothesis that a different incidence of apoptosis occurs in the mesenteric arteries of the spontaneously hypertensive rat (SHR) compared with its normotensive control the Wistar-Kyoto rat (WKY) at 1-2 weeks of age. DESIGN: We examined the incidence of apoptotic cells in the blood vessel wall of muscular arteries from the SHR and WKY at 1-2 weeks of age using two techniques of apoptosis measurement DNA laddering and 3'-OH end labelling. We also measured the volume of the blood vessel wall components and lumen sizes with the confocal microscope to determine whether a differential incidence of apoptosis occurred between the two rat strains. METHODS: We used phenol/chloroform extraction to isolate genomic DNA and assess DNA fragmentation, with gel electrophoresis to determine DNA laddering, and 3'-OH end labelling, where the enzyme terminal deoxynucleotidyl transferase catalyses the addition of fluorescein-conjugated nucleotides to the cut ends of DNA, to detect in situ DNA fragmentation. The volume per unit length of the blood vessel structural components was measured by optical sectioning with the confocal microscope. RESULTS: We found that the SHR had a significantly decreased incidence of cellular apoptosis over WKY. This was true for both the electrophoretic method where SHR had significantly less fragmented DNA (molecular size < 600 bp) than WKY (P= 0.01), and for the microscopic method where SHR had fewer labelled cells in both the adventitia (P= 0.01) and the media (P= 0.0001) layers of large mesenteric arteries. The volumes of the adventitia, media and lumen in the large mesenteric arteries were similar between the two strains at this age. CONCLUSION: These findings suggest that a differential incidence of cellular apoptosis at the age of 1 -2 weeks may be responsible for the larger media volume found in older SHR and thus contributes to the development of hypertension in these animals.

Perindopril treatment prolonged the lifespan of spontaneously hypertensive rats.

OBJECTIVE: The effects of perindopril treatment on hypertension development and the lifespan of adult spontaneously hypertensive rats (SHR) were studied. DESIGN: Adult male SHR (aged 15 weeks) were given once a day treatment with 4 mg/kg perindopril by gavage for 12 weeks. Littermates given distilled water were used as controls. The blood pressure and lifespan of these rats were studied. METHODS: The systolic blood pressure (SBP), heart rate and body weight of these rats were measured at regular intervals until they died from natural causes. At necropsy macroscopic and microscopic examinations were made of various organs to determine the cause of death. Serum levels of creatinine, urea and protein were also measured. RESULTS: Perindopril treatment resulted in the normalization of SBP after 2 weeks of treatment. Withdrawal of the treatment after 12 weeks of treatment caused an elevation of SBP, but the blood pressure of the treated SHR had remained in the normotensive range (< or = 150 mmHg). The heart rate and body weight of the SHR were not affected by the treatment. The average lifespan of the SHR was increased by 12 weeks compared with the control rats. The heart weight, brain lesions and arterial lesions were reduced by the treatment. CONCLUSION: A 12-week treatment of adult SHR with perindopril was effective in causing a permanent prevention of hypertension, amelioration of some of the tissue damage associated with hypertension and an increase in the lifespan of these rats.

Cross-over study comparing effects of treatment with an angiotensin converting enzyme inhibitor and an angiotensin II type 1 receptor antagonist on cardiovascular changes in hypertension.

OBJECTIVE: To compare the blood-pressure-lowering effects of an angiotensin converting enzyme inhibitor, perindopril, with those of an angiotensin II type 1 receptor antagonist, L-158,809, for adult spontaneously hypertensive rats. DESIGN: A cross-over design was used, to treat adult spontaneously hypertensive rats with one drug for 10 weeks, and then with the other for 5 weeks. METHODS: Adult, male spontaneously hypertensive rats (aged 15 weeks) were treated daily by gavage for 10 weeks with perindopril (P group) or L-158,809 (L group), then treatment was crossed over so that rats in the P group were treated with L-158,809 (P/L group) and rats in the L group were treated with perindopril (L/P group) for 5 weeks. Blood pressure was measured weekly. Plasma angiotensin converting enzyme activity, renal angiotensin receptor density, and arterial structure and functioning were measured after the single and crossover treatment periods. RESULTS: Treatment lowered the blood pressure from 206 +/- 2 mmHg in rats in the control group, to 126 +/- 2 in rats in the P group and 150 +/- 2 in rats in the L group. After the cross-over period, blood pressure decreased further from 150 +/- 2 to 129 +/- 3 mmHg in rats in the L/P group, whereas blood pressure of spontaneously hypertensive rats in the P/L group increased from 126 +/- 2 to 148 +/- 2 mmHg. Perindopril treatment almost abolished plasma angiotensin converting enzyme activity, whereas L-158,809 treatment had no effect. Renal angiotensin II receptor density was decreased versus baseline in rats in the P and L groups. The affinity of binding was decreased versus baseline in rats in the L group. A positive correlation to blood pressure was found for mesenteric artery wall thickness and wall: lumen ratio. Concentration for half-maximal effect for the response of mesenteric arteries from rats in the P group to norepinephrine was lower than that of the control group rats. Angiotensin II potentiated the norepinephrine-stimulated contraction of arteries from rats in the control and P groups, but not that of arteries from rats in the groups treated with L-158,809. CONCLUSION: Perindopril was more effective than was L-158,809 at lowering the blood pressure of adult spontaneously hypertensive rats, and at altering the structure and functioning of the arteries.