Abnormalities in growth characteristics of aortic smooth muscle cells in spontaneously hypertensive rats.

Comparative studies have shown that cultured vascular smooth muscle cells from spontaneously hypertensive rats (SHR) proliferate to a higher cell number, grow to a greater density, and have greater specific growth rate, particularly at a higher saturation density, than those of the normotensive Wistar-Kyoto (WKY) control rats. The growth difference was not due to varying cell survival nor to attachment ability after passage. The degree of DNA synthesis was estimated by [3H]thymidine incorporation into newly synthesized DNA. [3H]thymidine uptake increased with escalating concentrations of calf serum and reached a plateau at 5% calf serum in WKY rats, whereas an excessive, continuous rise was observed in SHR with up to a 20% concentration. [3H]thymidine incorporation into newly synthesized DNA was tested after stimulation by platelet-derived growth factor and epidermal growth factor. A significantly higher amount of newly synthesized DNA in vascular smooth muscle cells from SHR was noted when the cells were stimulated by platelet-derived growth factor or epidermal growth factor alone, and their simultaneous addition did not significantly change the 50% effective concentration but heightened the maximal response. These data provide evidence of increased aortic smooth muscle cell proliferation from aortas of SHR after mitogen stimulation and suggest a defect in growth stimulatory-inhibitory control.

Transforming growth factor beta 1 expression and effect in aortic smooth muscle cells from spontaneously hypertensive rats.

Previous studies demonstrated that in addition to an increased response to growth factors, cultured vascular smooth muscle cells derived from spontaneously hypertensive rats (SHRs) grow to a greater density than cells from normotensive Wistar-Kyoto (WKY) rats. Transforming growth factor beta 1 (TGF-beta 1) has a bimodal effect on vascular smooth muscle cell growth, depending on cell density. The present study investigated the relation between cell density and expression of the proto-oncogene c-fos and TGF-beta 1 in cells from WKY rats and SHRs. The results demonstrate an increased accumulation of c-fos mRNA in calf serum-stimulated SHR cells but only at a high cell density. The expression of TGF-beta 1 mRNA was enhanced in growing SHR cells at every density studied as early as 24 hours after inoculation, with a further increase at later times. The effect of exogenous TGF-beta 1 on new DNA synthesis was evaluated by [3H]thymidine incorporation. At a low cell density, TGF-beta 1 had no effect on DNA synthesis in either WKY or SHR vascular smooth muscle cells. At a high cell density, there was a significant increase of DNA synthesis in response to TGF-beta 1 in SHR cells without any effect in WKY cells. In conclusion, contact inhibition of vascular smooth muscle cells from SHRs at a higher cell density is accompanied by an earlier expression of the marker gene c-fos and preceded by an exaggerated expression of TGF-beta 1.(ABSTRACT TRUNCATED AT 250 WORDS)

Sertraline treatment of generalized social phobia: a 20-week, double-blind, placebo-controlled study.

OBJECTIVE: The authors evaluated the efficacy, safety, and tolerability of sertraline, a selective serotonin reuptake inhibitor, in the treatment of generalized social phobia. METHOD: Adult outpatients with generalized social phobia (N=204) from 10 Canadian centers were randomly assigned to receive sertraline or placebo in a 2:1 ratio for a 20-week double-blind study following a 1-week, single-blind, placebo run-in. The initial dose of sertraline was 50 mg/day with increases of 50 mg/day every 3 weeks permitted after the fourth week of treatment (dosing was flexible up to a maximum of 200 mg/day). Primary efficacy assessments were the percentage of patients rated much or very much improved on the Clinical Global Impression (CGI) improvement item and the mean changes from baseline to study endpoint in total score on the social phobia subscale of the Marks Fear Questionnaire and total score on the Brief Social Phobia Scale. RESULTS: In intent-to-treat endpoint analyses of 203 of the patients, significantly more of the 134 patients given sertraline (N=71 [53%]) than of the 69 patients receiving placebo (N=20 [29%]) were considered responders according to their CGI improvement scores at the end of treatment. The mean reductions in the social phobia subscale of the Marks Fear Questionnaire and in the total score on the Brief Social Phobia Scale were 32.6% and 34.3% in the sertraline group and 10.8% and 18.6% in the placebo group, respectively. Analysis of covariance showed superiority of sertraline over placebo on all primary and secondary efficacy measures. Sertraline was well tolerated: 103 (76%) of the 135 sertraline-treated patients and 54 (78%) of the 69 placebo-treated patients completed the study. CONCLUSIONS: Sertraline is an effective treatment for patients with generalized social phobia.

Characterization of 5-hydroxytryptamine1A properties of flesinoxan: in vivo electrophysiology and hypothermia study.

Flesinoxan is a high affinity and selective 5-hydroxytryptamine1A (5-HT1A) ligand which, unlike the 5-HT1A agonists of the azapirone class, does not generate 1-(2-pyrimidinyl)piperazine, an alpha 2-adrenoreceptor antagonist. In view of potential antidepressant effects of flesinoxan, this study was undertaken to characterize its 5-HT1A properties in the rat brain using in vivo electrophysiology and hypothermia paradigms. The suppressant effect of microiontophoretic applications of flesinoxan on the firing activity of CA3 pyramidal neurons was blocked by concomitant application of the 5-HT1A antagonist BMY 7378. Compared to gepirone, the efficacy of flesinoxan to suppress the firing activity of CA3 pyramidal neurons was significantly greater. While the coapplication of flesinoxan antagonized the suppressant effect of 5-HT on CA3 pyramidal neurons, it failed to do so on dorsal raphe 5-HT neurons, indicating that flesinoxan acts as a partial agonist at postsynaptic and as a full agonist at presynaptic 5-HT1A receptors. The capacity of flesinoxan to antagonize the effect of 5-HT on CA3 pyramidal neurons was similar to that of 8-hydroxy-2-(di-n-propylamino)-tetralin (8-OH-DPAT) and significantly greater than that of gepirone. The intravenous administration of flesinoxan suppressed the firing activity of both CA3 pyramidal neurons and dorsal raphe 5-HT neurons. However, when compared to 8-OH-DPAT, significantly higher doses of flesinoxan were required. The acute brain penetration of [3H]flesinoxan and [3H]8-OH-DPAT was, therefore, determined. Nine minutes after intravenous administration, [3H]8-OH-DPAT reached significantly greater brain concentration than [3H]flesinoxan. Subcutaneous administration of flesinoxan and 8-OH-DPAT produced a dose-dependent hypothermia. The flesinoxan-induced hypothermia was significantly attenuated by prior administration of the non-selective 5-HT1A antagonist pindolol and the 5-HT1/2 antagonist methysergide. Similar degrees of hypothermia were achieved with 3 mg/kg of flesinoxan and 0.5 mg/kg of 8-OH-DPAT. The maximal effect of flesinoxan occurred 30 min later than that of 8-OH-DPAT and faded more slowly. The 5-HT1A properties of flesinoxan suggest that it may be an effective anxiolytic/antidepressant agent.

Agonist occupation of serotonin1A receptors in the rat hippocampus prevents their inactivation by pertussis toxin.

The present study was undertaken to investigate the nature of the effect of pertussis toxin on the responsiveness of two potentially distinct subgroups of postsynaptic serotonin1A (5-HT1A) receptors of rat hippocampus CA3 pyramidal neurons: those located at the level of the cell body, which can be activated by microiontophoretically-applied 5-HT1A receptor agonists, and those located on dendrites, which can be activated by endogenous serotonin released by the stimulation of the ascending serotoninergic pathway. The former receptors (denoted as extrasynaptic) have been previously demonstrated to be sensitive to pertussis toxin, whereas the latter (denoted as intrasynaptic) have been shown to be pertussis toxin-insensitive. Rats treated with the 5-HT1A receptor agonists flesinoxan or BMY 42568 were used to determine whether tonic activation of extrasynaptic 5-HT1A receptors would prevent their inactivation by pertussis toxin. A pretreatment with p-chlorophenylalanine was used to determine whether a serotonin depletion would render the intrasynaptic 5-HT1A receptors sensitive to pertussis toxin. The responsiveness of CA3 pyramidal neurons to the suppressant effects of microiontophoretically-applied serotonin, 8-hydroxy-2-(di-n-propylamin)-tetralin, baclofen and GABA or to endogenously-released serotonin, elicited by the stimulation of the ascending serotoninergic pathway, was studied one to 10 days after the intrahippocampal injection of pertussis toxin. When compared to control saline-treated rats, the treatments with flesinoxan (5 mg/kg/day, s.c.) and BMY 42568 (5 mg/kg/day, s.c.) delivered for 14 days by osmotic minipumps, starting three days prior to the injection of pertussis toxin, significantly attenuated the effect of pertussis toxin on the responsiveness of CA3 pyramidal neurons to microiontophoretic applications of serotonin and 8-hydroxy-2-(di-n-propylamino)-tetralin, as well as baclofen, an agonist of GABAB receptors, which share the same G proteins with 5-HT1A receptors. The two-day pretreatment with p-chlorophenylalanine (350 mg/kg/day, i.p.) did not render the intrasynaptic 5-HT1A receptors sensitive to pertussis toxin, as indicated by the unchanged efficacy of the stimulation of the ascending serotonin pathway in the suppressing the firing activity of CA3 dorsal hippocampus pyramidal neurons. Our results suggest that the sustained activation of extrasynaptic 5-HT1A receptors prevents the pertussis toxin-induced ADP ribosylation of G protein alpha subunit, and thereby protects an amount of G proteins sufficient to maintain the function, not only of 5-HT1A, but also of GABAB receptors.(ABSTRACT TRUNCATED AT 400 WORDS)

[Intrinsic and extrinsic factors implicated in cell proliferation of vascular smooth muscle in hypertension and diabetes]. / Facteurs intrinsèques et extrinsèques impliqués dans la prolifération cellulaire de la musculature vasculaire lisse dans l'hypertension et le diabète.

The increased potential for growth of vascular smooth muscle cells in one of the key abnormalities in the development of essential hypertension, diabetic microangiopathy and atherosclerosis. The underlying mechanisms seem to be extrinsic (increased platelet-derived growth factor-like activity) and intrinsic (increased rate of growth, greater maximal response to growth factors and less contact inhibition). In this article, the authors discuss the primary role of an alteration in vascular smooth muscle cellular proliferation in hypertension, the extrinsic growth factors contained in platelet extracts of diabetic and hypertensive subjects and the specific effects of insulin and antihypertensive therapy on this pro-mitotic platelet activity. The result of experimental studies in our laboratory and in other studies suggest that genetic factors and therapeutic intervention could control the growth of vascular smooth muscle cells and that further evaluation of anti-hypertensive therapy may be necessary.

Vascular smooth muscle cell hyper-responsiveness to growth factors in hypertension.

To understand the intrinsic mechanisms of vascular smooth muscle cell proliferation, we studied the growth of cultured rat aortic smooth muscle from spontaneously hypertensive rats (SHR) and their normotensive controls, Wistar-Kyoto rats (WKY), under basal conditions and after stimulation. Cell growth was assessed by the determination of cell number, and incorporation of 3H-thymidine into newly-synthesized DNA. We demonstrated that vascular smooth muscle cells from SHR proliferate faster and grow to a greater density, regardless of the initial plating number. The growth difference was not due to different plating efficiencies. Significantly higher 3H-thymidine incorporation was observed in vascular smooth muscle cells from SHR when the cells were stimulated by calf serum, platelet-derived growth factor or epidermal growth factor. Exposure to calf serum elicited an excessive expression of c-myc and c-fos in the first hour after stimulation. These results provide evidence of the hyper-responsiveness of vascular smooth muscle cells from SHR aortae to growth stimuli.

Intrinsic factors involved in vascular smooth muscle cell proliferation in hypertension.

The exaggerated response to growth factors of vascular smooth muscle cells from spontaneously hypertensive rats when compared to cells from normotensive control Wistar-Kyoto rats persists in culture, indicating an intrinsic/genetic defect. The time course of 3H-thymidine incorporation shows that synchronized vascular smooth muscle cells from spontaneously hypertensive rats start to synthesize new DNA earlier after mitogenic stimulation than cells from normotensive rats. Flow cytometry demonstrates that in cell populations growing in 10% calf serum for three d there is a higher proportion of cells from spontaneously hypertensive rats in the S phase of the cell cycle. The same proportions in the G2 + M phase of growing, as well as synchronized cells from normotensive and hypertensive rats indicate no difference in polyploidy. Forward light scatter analysis reveals no difference in cell size. These results suggest that the growth kinetic of vascular smooth muscle cells from normotensive and spontaneously hypertensive rats are different. Since the defect seems to be in the prereplicative phase of the cell cycle susceptible to regulation by extrinsic factors, we studied the effect of the calmodulin inhibitor, W-7, on DNA synthesis. The comparable IC50 of W-7 to inhibit cell growth of vascular smooth muscle cells of both origins indicates that the defect may not be due only to calmodulin, and furthermore suggests the involvement of a previously-reported calmodulin activator in hypertension.

Accelerated entry of aortic smooth muscle cells from spontaneously hypertensive rats into the S phase of the cell cycle.

The present study was designed to characterize the growth kinetics of the exaggerated proliferative response to mitogens of vascular smooth muscle cells from spontaneously hypertensive rats compared with cells from normotensive Wistar-Kyoto controls. Cellular DNA content, analyzed by flow cytometry, demonstrated a 4-h accelerated entry into the S phase of the cell cycle of vascular smooth muscle cells from spontaneously hypertensive rats; the significant (4.5-fold) increase in the percentage of cells in the S phase occurred between 8 and 12 h after calf serum stimulation. A 3.9-fold increase of cells in the S phase was seen in the normotensive controls only between 12 and 16 h. Transit through the cell cycle was quantitated by flow cytometry using the Hoechst 33,342--bromodeoxyuridine substitution technique. Vascular smooth muscle cells from spontaneously hypertensive rats went through the cell cycle 4 h ahead of cells from normotensive Wistar-Kyoto rats. This accelerated transit of spontaneously hypertensive rat cells was mostly due to an earlier entry into the S phase. Persistence of this new intermediate phenotype in cell culture suggests its primary pathogenetic role in spontaneous hypertension.

Partial agonistic activity of R- and S-enantiomers of 8-OH-DPAT at 5-HT1A receptors.

In this study, the 5-HT1A agonistic activity of R- and S-enantiomers of the prototypical 5-HT1A agonist 8-OH-DPAT was investigated using in vivo microiontophoresis and the hypothermic response in rats. Both the R- and S-enantiomers suppressed current-dependently the firing activity of dorsal hippocampus CA3 pyramidal neurons. The number of spikes suppressed/nA of R-(+)-OH-DPAT was about 2-fold greater than that of S-(-)-OH-DPAT, which indicates greater agonistic activity of the R-enantiomer. The determination of the effectiveness of 5-HT in suppressing the firing activity of CA3 pyramidal neurons prior to and during application of either the R- or S-enantiomer showed that both compounds antagonized the effect of 5-HT, thus demonstrating their partial agonistic activity. Racemic 8-OH-DPAT produced a dose-dependent hypothermia which was attenuated by the 5-HT1A antagonist pindolol, but not by the nonselective 5-HT antagonist methysergide. Similarly, both R- and S-enantiomers induced a dose-dependent hypothermia, which was greater and longer lasting in the case of R-(+)-OH-DPAT when compared to S-(-)-OH-DPAT. In conclusion, R-(+)-OH-DPAT, displayed a greater agonistic activity at 5-HT1A receptors than S-(-)-OH-DPAT, both in suppressing firing activity of CA3 pyramidal neurons and in decreasing body temperature. Nevertheless, both compounds behaved as partial agonists.

Vascular smooth muscle cell proliferation and its therapeutic modulation in hypertension.

The increased growth potential of vascular smooth muscle cells (VSMCs) represents one of the crucial anomalies responsible for the development of essential hypertension, diabetic macroangiopathy, and atherosclerosis. The exaggerated response to growth factors of VSMC from spontaneously hypertensive rats (SHRs) persists in culture when compared with normotensive Wistar-Kyoto control rats, indicating an intrinsic defect in the hypertension-producing mechanism. This greater proliferation is characterized by two intermediate phenotypes: (1) accelerated entry into the S phase of the cell cycle, which results from hyperresponsiveness to epidermal growth factor and platelet-derived growth factor, and (2) abnormal contact inhibition. The enhanced expression of transforming growth factor beta 1 (TGF-beta 1) messenger ribonucleic acid in SHRs precedes this altered contact inhibition, and only VSMCs from SHRs respond to exogenously added TGF-beta 1 at a high cell density, which suggests that abnormal TGF-beta 1 autoregulation may be implicated in the second phenotype. Platelets contain major growth factors for VSMC. Platelet extracts from hypertensive and diabetic patients present augmented growth-promoting activity on VSMCs, which is most evident when both diseases occur simultaneously. Growth-promoting activity may be further influenced by antihypertensive therapy. This growth-promoting activity is increased by hydrochlorothiazide but not by indapamide, atenolol, or captopril in diabetic hypertensive and nondiabetic hypertensive patients. In conclusion, VSMCs in hypertension manifest an intrinsic growth defect that is modulated by extrinsic platelet growth factors and antihypertensive drugs.

Enhanced growth-dependent expression of TGF beta 1 and hsp70 genes in aortic smooth muscle cells from spontaneously hypertensive rats.

Enhanced proliferation of vascular smooth muscle cells (VSMC) from spontaneously hypertensive rats (SHR) as compared with Wistar-Kyoto rats (WKY) persists in long-term culture and is characterized by an accelerated entry of these cells into the synthetic S phase of the cell cycle and a higher specific growth rate, particularly evident at high cell density. In the present study, we investigated by Northern blot experiments the expression of genes putatively involved in the regulation of VSMC growth. One of them is the transforming growth factor beta 1 gene (TGF beta 1), a bifunctional modulator of cell growth whose action is dependent on cell density. The accumulation of TGF beta 1 mRNA was enhanced in growing SHR cells at every density studied as early as 24 h after inoculation with a further increase at later times. Protooncogenes c-fos and c-myc, which have been implicated in G1/S phase transition, have also been investigated in VSMC by Northern blot analysis. At low cell density, calf serum stimulated c-fos and c-myc mRNA expression was comparable in WKY and SHR cells whereas at high cell density, c-fos induction was higher in VSMC from SHR. SHR VSMC respond more to mitogenic stimulation and to environmental (e.g., heat) stress, particularly when growing near saturation density. hsp70 constitutes a gene family responsive to environmental stimuli (heat) and to mitogenic stimulation.(ABSTRACT TRUNCATED AT 250 WORDS)

Prevention of relapse in generalized social phobia: results of a 24-week study in responders to 20 weeks of sertraline treatment.

The aim of this study was to evaluate the efficacy, tolerability, and effects on quality of life of sertraline, a selective serotonin reuptake inhibitor, in the prevention of relapse of generalized social phobia. Fifty adult outpatients with generalized social phobia who were rated much or very much improved on the Clinical Global Impression Scale of Improvement (CGI-I) after 20 weeks of sertraline treatment (50-200 mg/day) were randomly assigned in a one-to-one ratio to either continue double-blind treatment with sertraline or immediately switch to placebo for another 24 weeks. The initial 20-week study was placebo-controlled, and 15 responders to placebo also continued to receive double-blind placebo treatment in the continuation study. Eighty-eight percent of patients in the sertraline-continuation group and only 40% of patients in the placebo-switch and placebo-responder groups completed the study. In intent-to-treat endpoint analyses, 1 (4%) of 25 patients in the sertraline-continuation group and 9 (36%) of 25 patients in the placebo-switch group had relapsed at study endpoint (chi2 = 8.0, Fisher exact test, p = 0.01). The relative risk (hazards ratio) for relapse associated with placebo-switch relative to sertraline-continuation treatment was 10.2 (95% confidence interval, 1.3-80.7). Mean CGI-Severity, Marks Fear Questionnaire (MFQ) Social Phobia subscale, and Duke Brief Social Phobia Scale (BSPS) total scores were reduced by 0.07, 0.34, and 1.86 in the Sertraline-Continuation group and increased by 0.88, 4.09, and 5.99 in the Placebo-Switch group (all F > 5.3, p < 0.03), respectively. CGI-Severity, MFQ Social Phobia subscale, and BSPS scores also increased in the Placebo-Responder group. Discontinuations because of lack of efficacy were 4% in the sertraline-continuation group, 28% in the placebo-switch group (chi2 = 5.36, Fisher exact test, p = 0.049), relative to sertraline, and 27% in the placebo-responder group. Sertraline was effective in preventing relapse of generalized social phobia. Future research should assess whether improvements may be maintained or further increased by longer periods of treatment or through the addition of cognitive-behavioral techniques.