Insulin has been shown to attenuate pressor-induced vascular contraction, but the mechanism for this vasodilatory action is unknown. This study examines the effect of insulin on angiotensin II (ANG II)-induced increments in cytosolic calcium in cultured rat vascular smooth muscle cells (VSMC). 20-min incubations with insulin (10 ,U/ml to 100 mU/ml) did not alter basal intracellular calcium concentration (ICa2I,), but inhibited the response to 100 nM ANGII in a dose-dependent manner (ANG II alone, 721±54 vs. ANG II + 100 mU/ml insulin, 315±35 nM, P < 0.01). A similar effect of insulin on ANGII action was observed in calcium poor buffer. Moreover, insulin did not effect calcium influx. ANG II receptor density and affinity were not affected by 24-h incubation with insulin. To further clarify the mechanisms of these observations, we measured ANG II-induced production of inositol 1,4,5-triphosphate (IP3), and IP3-releasable 45Ca. Insulin treatment did not alter ANG Il-stimulated IP3 production. However, IP3-stimulated release of45Ca in digitonin permeabilized cells was significantly reduced after 5-min incubations with 100 mU/ml insulin. Thapsigargin induced release of calcium stores was also blocked by insulin. Thus, insulin attenuates ANG 11-stimulated ICa2"I, primarily by altering IP3-releasable calcium stores. Insulin effects on ANG II-induced ICa2`Ii were mimicked by preincubation of VSMC with either sodium nitroprusside or 8-bromo-cGMP. As elevations in cGMP in vascular tissue lower ICa2I1j, it is possible that insulin affects IP3 release of calcium by a cGMP-dependent mechanism that would contribute to its vasodilatory effects. (J. Clin. Invest. 1993. 92:1161-1167

This study was conducted to further investigate angiotensinogen synthesis in rat aortic smooth muscle cells (SMC) grown in culture. tissue cultures maintained in defined medium neither grew nor synthesized angiotensinogen. However, in the presence of 5% homologous serum both cell proliferation and angiotensinogen synthesis became apparent. Substitution of normal control serum with that of bilaterally nephrectomized rats or animals given dexamethasone (10mg/kg, ip) led to a further significant increase in angiotensinogen production. In contrast, serum from adrenalectomized rats suppressed angiotensinogen synthesis below the rate observed with normal serum. A positive linear correlation (r = 0.96, p less than 0.01) was evident between the serum angiotensinogen level and the rate of de novo synthesis of this protein. No correlations were found between cell proliferation and either angiotensinogen synthesis or serum angiotensinogen levels. Dexamethasone added to serum did not stimulate the rate of angiotensinogen synthesis and appeared to inhibit cell proliferation. Stimulation or suppression of angiotensinogen synthesis was not accompanied by a statistically significant change in angiotensinogen specific mRNA. The data indicate a complex regulation of angiotensinogen in vascular smooth muscle cells in culture.

Cells that expressed the muscle-specific intermediate filament protein desmin were cultured from the aorta of Fischer 344 rats. When the cultured cells were extracted with digitonin, they accumulated 45Ca2+ from the incubation medium in a manner that was stimulated by ATP and released subsequently by exposure to the Ca2+ ionophore A23187. Ca2+ bound in the presence of ATP was also released by exposure to inositol 1,4,5-trisphosphate (IP3). Like contraction in some kinds of smooth muscle, IP3 released Ca2+ in either the absence or the presence of the ATPase-inhibitor ruthenium red. When the responsiveness of digitonin-extracted cells cultured from 3-, 12-, and 24-month-old rats was compared, cells from the youngest group released only about one-half as much Ca2+ as cells from the 12- or 24-month-old rats. The results suggest that in the rat there are changes during maturation in the responsiveness to inositol polyphosphates of intracellular compartments that sequester Ca2+ for stimulus-contraction coupling in the aortic smooth muscle cell. These changes, characterized in smooth muscle cells in vitro, might contribute to the way vascular responsiveness is regulated in vivo.

Parathyroid hormone and parathyroid hormonerelated protein lower blood pressure and relax contracted arteries. Parathyroid hormone also attenuates angiotensin H-induced vasoconstriction. To determine the cellular mechanism or mechanisms by which parathyroid hormone analogues antagonize pressor effects, we examined the effect of these peptides on angiotensin II-induced calcium mobilization in fura 2-AMloaded cultured rat vascular smooth muscle cells. Either 100 nmol/L parathyroid hormone or parathyroid hormone-related protein significantly reduced the amount of calcium mobilized by 100 nmol/L angiotensin II. The attenuating effect of these peptides was mimicked by 10 mmol/L forskolin and 10 mmol/L isobutylmethylxanthine and was not dependent on the presence of extracellular calcium. This effect of the parathyroid hormone analogues was reduced when cells were pretreated with 100 mmol/L 2',5'-dideoxyadenosine, an adenylate cyclase inhibitor. Combined inhibition of cyclic nucleotide-dependent protein N umerous studies have consistently demonstrated that parathyroid hormone (PTH), as either intact hormone or the N-terminal 1-34 fragment of PTH [PTH(l-34)], relaxes contracted vessels and lowers blood pressure in a variety of animal species.15 PTH can also attenuate vasopressor responses to angiotensin II (Ang II) and norepinephrine when coinfused with these pressors in rats. 6 Although initially identified as a factor in the generation of hypercalcemia in a variety of malignancies, PTH-related protein (PTHrp) has subsequently been found to be present in a variety of normal tissue, including vascular smooth muscle. 7 The N-terminal fragment of this hormone, PTHrp(l-34), appears to affect target cells much like PTH(l-34) does and appears to bind to the same receptor or receptors as PTH(l-34) in bone, kidney, and renal vascular cells. 89Acute exposure to PTHrp(l-34) has been shown to cause vasodilation in vivo, 10 and it has been suggested that PTHrp may have an autocrine or paracrine function in modulating blood flow in certain tissues such as myometrium. 11Although it has been established that PTH and PTHrp acutely decrease blood pressure and relax vasReceived June 14,1993; accepted in revised form December 13, 1993.From the Department of Endocrinology, Veterans Affairs Medical Center, Sepulveda, and the UCLA School of Medicine, Los Angeles, Calif.Correspondence to Arnold S. Brickman, MD, Endocrinology Department (HIE), Sepulveda VAMC, 16111 Plummer St, Sepulveda, CA 91343. kinases eliminated the inhibitory effect of parathyroid hormone, whereas protein kinase C inhibition had no effect. Parathyroid hormone analogues decreased the amount of calcium released by inositol 1,4,5 -trisphosphate in digitoninpermeabilized vascular smooth muscle cells. This effect was inhibited by treatment with 2',5'-dideoxyadenosine. These results suggest that these peptides attenuate inositol 1,4,5-trisphosphate-sensitive calcium mobilized by angiotensin II via an adenylate cyclase-dependent mechanism. This may be a mechanism ...

Smooth muscle-mediated expansion and contraction of the vascular sinusoids of the corpora cavernosa may modulate male erectile function. To elucidate the biochemical events that control erection by promoting or inhibiting contraction of cavernosal smooth muscle, tissue from a potent man was grown in cell culture. The cells grew as noncontractile cultures, but had the following smooth muscle cell properties: These cells expressed desmin, the muscle cell-specific intermediate filament protein. They accumulated 45Ca2+ from the medium, which was released by exposure to the ionophore A23187, to cyclic nucleotides (cyclic guanosine 5'-monophosphate [GMP] much greater than cyclic adenosine 3',5'-monophosphate [AMP]), and to the phosphodiesterase inhibitor, papaverine; and; they accumulated Ca2+ in an ATP-dependent manner when the cultured cells were permeabilized by digitonin extraction. ATP-dependent Ca2+ uptake was inhibited approximately 80% by ruthenium red and simulated by cyclic GMP much greater than cyclic AMP. Inositol 1,4,5-trisphosphate (IP3), which is thought to mediate the release of Ca2+ by the smooth muscle cell sarcoplasmic reticulum in vivo, released approximately 0.85 pmol Ca2+/million cells from the digitonin-extracted cells. IP3-dependent release occurred in the presence of ruthenium red and was not affected by cyclic GMP or cyclic AMP. These results indicate that smooth muscle from this human source can be grown successfully in cell culture and that the biochemical pathways that regulate tension in vivo may be perpetuated in vitro. Moreover, some of the clinical responses to drugs administered in situ for erectile dysfunction (e.g. papaverine) may be the result of altered cavernosal smooth muscle cell Ca2+ exchange and may be mediated by cyclic GMP.