Previous studies have shown that 17 beta-estradiol (beta-E2) has a direct acute inhibitory effect on vascular smooth muscle (VSM) contraction. To investigate the mechanisms underlying this phenomenon, we utilized whole cell patch-clamping techniques to study effects of beta-E2 on voltage-dependent Ca2+ channels in cultured VSM cells (VSMC). T- and L-type Ca2+ currents were characterized with ramp and pulse protocols in A7r5 cultured VSMC. T-type current, inactivated in < 100 ms, was reduced by Ba2+ and was comparatively little affected by isradipine. L-type current required higher voltages to activate, inactivated slowly, was greatly increased by Ba2+, and could be completely inhibited by 5 microM isradipine. beta-E2 (10 microM) significantly reduced peak L-type Ba2+ current and T-type Ca2+ current within 1-2 min, whereas alpha E2 (a hormonally inactive isomer of estradiol) caused significantly less reduction in both types of current. Vehicle (0.1% ethanol) had no significant effect on either current. The inhibitory effect of beta-E2 on voltage-dependent Ca2+ currents may contribute to previously demonstrated beta-E2 attenuation of VSM contraction.

Pioglitazone, an insulin-sensitizing, antidiabetic agent, has blood pressure-lowering effects in insulin-resistant hypertensive rats and attenuates growth factor-induced increases of intracellular Ca 2+ in rat aortic vascular smooth muscle cells. To determine whether modulation of voltagedependent Ca 2+ channels plays a role in this association, we investigated the effects of pioglitazone on voltage-dependent current in cultured rat aortic (a7r5) and freshly dissociated rat tail artery vascular smooth muscle cells. Both cell types were studied with whole-cell patch-clamp techniques. Current through L-type Ca 2+ channels was elicited with a voltage ramp in the presence of Ba 2+ substituted for Ca 2+ . T-type Ca 2+ current was studied using a two-pulse protocol that enabled the isolation of transient current. In a7r5 vascular smooth muscle cells, 2-minute application of pioglitazone (5 and 10 /imol/L) reduced L-type current by 7.9±1.0% (n=8)

We investigated the effects of cyclic stretch on vascular smooth muscle cell (VSMC) alignment and potential overlap of signaling modalities with stretch-induced proliferation. VSMC were subjected to graded stretch (1 Hz at 100-124% of resting length) for 48 h. Graded stretch resulted in graded VSMC alignment from a minimum of completely random orientation to a maximum of ~80-85 degrees to the stretch vector. Alignment was reversible within 48 h of stretch cessation and independent of signaling modalities mediating stretch-induced proliferation: modulation of IGF-1, MAPK, phosphatidylinositol 3-kinase, tyrosine kinase, and stretch-activated calcium channels did not affect alignment. Nitric oxide (NO) synthase (NOS) blockade uncoupled alignment. Neither the NO donor, cytokine-induced NOS activity, nor L-citrulline affected alignment, but inhibited VSMC proliferation. Therefore, stretch-induced proliferation and alignment are differentially regulated, with NO a common signaling molecule for both. Targeting NOS in states such as restenosis and hypertension may prove to be beneficial.

Insulin attenuates the contractile responses of vascular smooth muscle (VSM) to various agonists. Insulinopenic and insulinresistant rats lack this normal attenuation of vascular contractile responses. To study this attenuating mechanism, the effects of insulin on calcium (Ca2") responses of cultured VSM cells (a7r5) to arginine vasopressin (AVP) and membrane potential were investigated. Insulin (1 and 100 mU/ml) shifted AVP dose-response curves to the right, reducing relative potency of AVP by 16-fold and 220-fold, respectively. Responses to AVP were significantly attenuated within 30 min of insulin application. The AVP-elicited rise in [Ca2"I] was partially dependent upon extracellular Ca2". AVP-elicited inward current was reduced by 90 min of insulin treatment (100 mU/ml), from a peak current of -103±27 pA (normal) to -37±15 pA (insulin treated). Peak voltage-dependent Ca2"-dependent inward current was unaffected by insulin; however, the current-voltage curve was shifted 16±3 mV to the right by insulin. Thus, insulin may reduce VSM contractile responses by attenuating agonistmediated rises in ICa2"I mediated, in part, by reductions in Ca2" influx through both receptor-and voltage-operated channels. (J. Clin. Invest. 1991. 88:1230-1236

Objective In this study we modeled repetitive motion strain (RMS) and myofascial release (MFR) in vitro to investigate possible cellular and molecular mechanisms to potentially explain the immediate clinical outcomes associated with RMS and MFR. Method Cultured human fibroblasts were strained with 8 hours RMS, 60 seconds MFR and combined treatment; RMS+MFR. Fibroblasts were immediately sampled upon cessation of strain and evaluated for cell morphology, cytokine secretions, proliferation, apoptosis, and potential changes to intracellular signaling molecules. Results RMS induced fibroblast elongation of lameopodia, cellular decentralization, reduction of cell to cell contact and significant decreases in cell area to perimeter ratios compared to all other experimental groups (p<0.0001). Cellular proliferation indicated no change among any treatment group; however RMS resulted in a significant increase in apoptosis rate (p<0.05) along with increases in death-associated protein kinase (DAPK) and focal adhesion kinase (FAK) phosphorylation by 74% and 58% respectively, when compared to control. These responses were not observed in the MFR and RMS+MFR group. Of the twenty cytokines measured there was a significant increase in GRO secretion in the RMS+MFR group when compared to control and MFR alone. Conclusion Our modeled injury (RMS) appropriately displayed enhanced apoptosis activity and loss of intercellular integrity that is consistent with pro-apoptotic DAPK2 and FAK signaling. Treatment with MFR following RMS resulted in normalization in apoptotic rate and cell morphology both consistent with changes observed in DAPK2. These in vitro studies build upon the cellular evidence base needed to fully explain clinical efficacy of manual manipulative therapies.