Jie Shan

Vascular Effects of Progesterone: Role of Cellular Calcium Regulation

Vascular actions of progesterone have been reported, independently of estrogen, affecting both blood pressure and other aspects of the cardiovascular system. To study possible mechanisms underlying these effects, we examined the effects of P in vivo in intact rats and in vitro in isolated artery and vascular smooth muscle cell preparations. In anesthetized Sprague-Dawley rats , bolus intravenous injections of P (100 &mgr;g/kg) significantly decreased pressor responses to norepinephrine (0.3 &mgr;g/kg). In vitro, progesterone (10−8 to 10−5 mmol/L) produced a significant, dose-dependent relaxation of isolated helical strips, both of rat tail artery precontracted with KCl (60 mmol/L) or arginine vasopressin (3 nmol/L), and of rat aorta precontracted with KCl (60 mmol/L) or norepinephrine (0.1 &mgr;mol/L). In isolated vascular smooth muscle cells, progesterone (5×10−7 mol/L) reversibly inhibited KCl (30 mmol/L) -induced elevation of cytosolic-free calcium by 64.1±5.5% (P <0.05), and in whole-cell patch-clamp experiments, progesterone (5×10−6 mol/L) reversibly and significantly blunted L-type calcium channel inward current, decreasing peak inward current to 65.7±4.3% of the control value (P <0.05). Our results provide evidence that progesterone is a vasoactive hormone, inhibiting agonist-induced vasoconstriction. The data further suggest that progesterone effects on vascular tissue may, at least in part, be mediated by modulation of the L-type calcium channel current activity and, consequently, of cytosolic-free calcium content.

Opioid receptor agonistic characteristics of mitragynine pseudoindoxyl in comparison with mitragynine derived from Thai medicinal plant Mitragyna speciosa

We have previously elucidated the opiate-like action of mitragynine, an active principle isolated from the Thai medicinal plant Mitragyna speciosa. In the present study, effects of the related compound, mitragynine pseudoindoxyl on electrically stimulated contraction in guinea pig ileum and mouse vas deferens, and on its binding affinity in the guinea pig brain membranes were studied. Mitragynine pseudoindoxyl inhibited the electrically stimulated ileum and mouse vas deferens contractions in a concentration-dependent manner. In the ileum, the effective concentration is in an nM order, being nearly equivalent to reported concentrations of the micro-opioid receptor agonist [D-Ala2, Met-Phe4, Gly-ol5] enkephalin (DAMGO), and is 100- and 20-fold smaller than those of mitragynine and morphine, respectively. In the vas deferens, it is 35-fold smaller than that of morphine. The inhibitory action of mitragynine pseudoindoxyl in the ileum was antagonized by the non-selective opioid receptor antagonist naloxone and the micro-receptor antagonist naloxonazine. It was also antagonized by the delta-receptor antagonist naltrindole in the vas deferens. Mitragynine pseudoindoxyl showed a similar binding affinity to DAMGO and naltrindole at micro- and delta-receptors, respectively. However, the affinity at kappa-receptors was negligible. The present study demonstrates that mitragynine pseudoindoxyl, a novel alkaloid structurally different from other opioid agonists, acts on opioid receptors, leading to a potent inhibition of electrically stimulated contraction in the ileum through the micro-receptors and in mouse vas deferens through delta-receptors.

Effects of Dehydroepiandrosterone Sulfate on Cellular Calcium Responsiveness and Vascular Contractility

Dehydroepiandrosterone sulfate (DHEAS) is an endogenous steroid having a wide variety of biological effects, but its physiological role remains undefined. Since an age-related decline of DHEAS corresponds to the progressive onset of atherosclerosis, cardiovascular diseases, and overall mortality, we investigated a possible protective role of DHEAS in vascular disease by studying the effects of this hormone (10(-7) to 10(-5) mol/L) on cytosolic free calcium and contractility in different in vitro vascular tissue preparations. DHEAS produced a significant, dose-dependent relaxation of isolated helical strips of rat tail artery precontracted with KCl (60 mmol/L) (89.7 +/- 18.7%, P < .01), arginine vasopressin (3 nmol/L) (27.3 +/- 7.1%, P < .01), and norepinephrine (0.1 mumol/L) (49.2 +/- 18.2%, P < .01). In isolated vascular smooth muscle cells DHEAS reversibly inhibited KCl (30 mmol/L)-induced elevations of cytosolic free calcium to 69.8 +/- 8.4% and 43.8 +/- 7.4% of the control response at 5 x 10(-7) and 5 x 10(-6) mol/L, respectively (P < .05 at both doses). These results provide evidence of a direct vascular action of DHEAS, in doses reflecting circulating levels in vivo, and suggest the possibility that these effects are mediated by modulation of intracellular calcium metabolism. We hypothesize that physiologically, DHEAS may serve to buffer vascular responsiveness to a wide variety of depolarizing and constrictor hormonal stimuli.

Purification and structural characterization of parathyroid hypertensive factor.

Parathyroid hypertensive factor (PHF) has been purified from two sources of material: plasma of spontaneously hypertensive rats (SHRs) and culture medium from organ culture of SHR parathyroid glands. Chromatographic characteristics of PHF from these two sources are identical. Biological activity of PHF (assayed as the characteristic delayed hypertensive response in normotensive rats) is sensitive to degradation by treatment in base, and the enzymes trypsin, chymotrypsin, phospholipase C, and phospholipase D. PHF activity may also be extracted from source material with chloroform: methanol (4:1). A hypothetical structure for the active component of PHF is suggested. This is comprised of a peptide linked to a lysophospholipid.

PHF: The New Parathyroid Hypertensive Factor

Parathyroid Hypertensive Factor (PHF) was discovered in SHR rats as a circulating substance with a unique delayed (60-90 min) hypertensive effect when injected into a normotensive assay rat. Subsequently, this correlation with hypertension was established in humans, especially in low-renin, salt-sensitive patients. Animal model studies also confirmed this correlation. Endocrinectomy and glandular replacement studies suggested that the parathyroid gland was the source of PHF. Subsequently, glands and cells in culture were also shown to secrete the substance. Other studies verified the parathyroid origin of PHF. The mechanism of action of PHF was shown to rely mainly on the opening of L-type calcium channels in vascular smooth muscle cells with an increase in [Ca2++]i. It is known that diseases other than hypertension often show increased [Ca2++]i and clinical features similar to hypertension, among them Type II diabetes. A recent study shows a correlation between circulating PHF level and Type II diabetes irrespective of the blood pressure status of the patient. It is suggested that PHF may be a [Ca++]i modulator, an excessive amount of which in the circulation may act on various target tissues, resulting in various disease symptoms with hypertension as an example. There may be many other such PHF-related diseases yet to be identified.

Mechanism of the Vascular Action of Parathyroid Hypertensive Factor

The present studies investigated the effect of parathyroid hypertensive factor (PHF) on intracellular calcium regulation in VSMC. Nifedipine inhibited the hypertensive effect of PHF in Sprague-Dawley (SD) rats in vivo. PHF amplified the L-type calcium current in vascular smooth-muscle cells (VSMCs) isolated from SD rat tail artery. PHF potentiated the tension induced by norepinephrine (NE) in the presence of normal added CaCl2 and inhibited the tension dependent on Ca2+ release from intracellular calcium store(s) induced by NE in SD rat tail artery helical strips. PHF potentiated the intracellular free calcium concentration ([Ca2+]i) increment induced by KCl in cultured VSMCs from SD rat tail artery. All of the in vitro cellular calcium effects of PHF temporally correlated with its delayed hypertensive effect in vivo. PHF did not affect the accumulation of inositolphosphates in SD rat tail artery. Infusion of theophylline blunted the hypertensive effect of PHF in SD rats, suggesting that PHF may stimulate phosphodiesterase (PDE) activity. We suggest that PHF may potentiate the effects of other vasoconstrictors on calcium channels and increase [Ca2+]i, which would then lead to an increase in the responsiveness of the VSMC to other vasoconstrictors, and therefore an increase in blood pressure. The action of PHF may involve stimulation of PDE activity.

Control of calcium channels in neuroblastoma cells (N1E-115)

Neuroblastoma cells (N1E-115) were used as models of transient (T) and long-lasting (L) Ca++ channels. The whole cell version of the patch clamp technique was used to measure inward Ca++ currents, and the fluorescent indicator, Fura-2, was used to measure changes in intracellular Ca++. Cells were cultured and selected during recording so that predominantly T or L channel currents were measured. T channel currents did not respond to dihydropyridine or parathyroid hormone, whereas L channel currents did. BAY-K-8644 increased and nifedipine decreased L channel currents. After a 15 mM KCl challenge, cells with predominantly T channels responded with a transient change in intracellular Ca++, while cells with predominantly L channels showed a sustained response. PTH inhibited the increase in intracellular Ca++ in cells with L channels, but not in those with T channels. PTH may be an example of an endogenous calcium channel blocker, at least in neuroblastoma cells.

Clinical aspects of parathyroid hypertensive factor.

To determine the clinical significance of parathyroid hypertensive factor (PHF), physiological studies previously performed in animal models of hypertension were parallelled by human studies. These studies revealed that PHF-like activity is present in human hypertension, where it correlates with the salt-sensitive, low-renin state. As in spontaneously hypertensive rats, both supplemental calcium and calcium-channel blockers appear to be useful in the treatment of PHF-related hypertension. In primary hyper-parathyroid patients, PHF presence is linked with the presence of hypertension. Postparathyroidectomy blood pressure falls in parallel with PHF levels. These preliminary human studies suggest that PHF may be a useful marker in the treatment of hypertension.