Kent Hermsmeyer

Percutaneous absorption of progesterone in postmenopausal women treated with transdermal estrogen.

OBJECTIVE The objective of this study was to evaluate the serum levels of progesterone resulting from the application of a progesterone cream to the skin. STUDY DESIGN Six postmenopausal women were evaluated at a university clinic over a 4-week period. RESULTS Transdermal estradiol 0.05 mg was applied 2 days before the first application of progesterone (30 mg/d) and was continued throughout the study. Patches were changed twice a week. Progesterone cream was applied once a day for 2 weeks. On day 15 and for the next 2 weeks, the progesterone cream was applied twice daily (60 mg/d). Serum 17beta-estradiol and progesterone were measured at 9 different times over a 24-hour period on day 1 and at weekly intervals for the 4-week duration of the study. Serum 17beta-estradiol concentrations varied among women, with mean concentrations of 40 to 64 pg/mL observed. Consistency in 17beta-estradiol concentrations was found within individual persons throughout the study. Serum progesterone concentrations also varied among women, with mean concentrations ranging from 1.6 to 3.3 ng/mL. After 2 weeks of percutaneous dosing, progesterone concentrations were sustained for at least 8 hours and were consistent within a given person. An appropriate increase in progesterone concentration occurred after 4 weeks compared with 2 weeks of application. Individually, a 0.53 correlation, significant at P <.0001, was seen between the absorption of 17beta-estradiol and progesterone. CONCLUSION Significant increases in serum concentrations of progesterone were observed in all of the women studied. The percutaneous absorption of progesterone correlates strongly with the absorption of transdermal 17beta-estradiol. There is variance in absorption of progesterone just as with 17beta-estradiol, and the 2 measures are closely correlated. The percutaneous application of progesterone cream appears to be a safe and effective route of administration.

In vitro modulation of primate coronary vascular muscle cell reactivity by ovarian steroid hormones

Susceptibility to drug‐induced coronary vasospasm in rhesus monkeys increases after removal of the ovaries and can be normalized by adding back physiological levels of estradiol‐17β (E2) and/or natural progesterone (P) in vivo as reported recently by our group. Furthermore, the reactivity status (Ca2+ and protein kinase C responses) of freshly isolated and primary culture coronary artery vascular muscle cells (VMC) mimic the intact coronary artery responses to 5‐HT + U46619. Since coronary reactivity is maintained in the isolated VMC, we hypothesized that the reactivity state inherent in the VMC was modulated directly by ovarian steroids in vitro as in the whole animal. To test this hypothesis, we treated hyperreactive VMC from ovariectomized (ovx) monkeys in vitro with E2 or P and measured VMC reactivity to combined stimulation with 5‐HT and U46619, as determined by the amplitude and especially the duration of intracellular Ca2+ signals, as well as protein kinase C (PKC) activation/translocation. VMC were treated for 12–96 h with 3–100 pg/ml E2 (10–365 pM) and/or 0.3–3 ng/ml P (0.95–9.5 nM). Hyperreactive responses to the combination of 5‐HT and U46619 in untreated VMC were significantly and dose‐dependently reduced by treatment in vitro with physiological levels of either E2 or P for at least 24 h. Both the early transient and late sustained increases in intracellular Ca2+ and PKC translocation were blunted, and the effects of 0.2 nM E2 and 3.2 nM P were specifically antagonized by the receptor blockers ICI 182,780 (200 nM) and RU486 (15 nM), respectively. Antibodies to the estrogen receptor and progesterone receptor labeled nuclei in VMC, which were also positively labeled by a smooth muscle myosin heavy chain monoclonal antibody. These data indicate that natural ovarian steroids directly reduce hyperreactive 5‐HT and thromboxane A2‐stimulated Ca2+ and PKC responses of coronary artery VMC from surgically menopausal rhesus macaques. We hypothesize that vascular hyperreactivity, which may be a critical factor involved in the increased incidence of coronary artery vasospasm and ischemic heart disease in postmenopausal women, can be normalized by E2 and/or P through direct actions on coronary artery vascular muscle cells.—Minshall, R. D., Miyagawa, K., Chadwick, C. C., Novy,M. J., Hermsmeyer, K. In vitro modulation of primate coronary vascular muscle cell reactivity by ovarian steroid hormones. FASEB J. 12, 1419–1429 (1998)

Physiologic and pathophysiologic relevance of T-type calcium-ion channels: potential indications for T-type calcium antagonists.

The family of voltage-gated calcium-ion (Ca2+) channels is critical in the role of transmembrane signaling of excitable cells throughout the body. Within the cardiovascular system, two types of Ca2+ channels have been identified: the L-type channel and the T-type channel. These two types of Ca2+ channels have distinct electrophysiologic identities, and although the roles of the T-type Ca2+ channels have not been firmly established, there are many reasons for believing that the roles of the T-type and L-type Ca2+ channels are distinct. T-type Ca2+ channels have the appropriate characteristics to generate pacemaker activity in the sinoatrial node. In vascular smooth muscle, they appear to be involved in maintenance of coronary and peripheral vasomotor tone and control of vascular growth and remodeling. Characterization of the T-type Ca2+ channels will be facilitated by the availability of mibefradil, a novel calcium antagonist that selectively blocks T-type Ca2+ channels. Mibefradil is associated with a reduction in heart rate but not with negative inotropic effects or neurohormonal stimulation. It is thought that the unique pharmacologic effects of mibefradil are related to blockade of T-type Ca2+ channels, and it is hypothesized that this action will have a positive impact on cardiovascular morbidity and mortality via cardioprotective and renoprotective effects. However, much work needs to be done to fully test this hypothesis.

Stroke-Prone SHR Vascular Muscle Ca2+ Current Amplitudes Correlate with Lethal Increases in Blood Pressure

Studies on the possible causal relationship between the Ca2+ channel current density in the vascular muscle cell (VMC) and increases in blood pressure were extended by a comparison of stroke-prone spontaneously hypertensive rats (SP-SHR) with N/nih outbred normotensive rats. Maximal amplitudes of both L-type and T-type Ca2+ channel currents were significantly increased in SP-SHR without a difference in cell capacitance. SP-SHR peak current amplitudes in 20 mM Ba2+ averaged 446 +/- 64 pA while N/nih averaged 156 +/- 25 pA (clearly separated statistically). Both L-type and T-type Ba2+ currents (IBa) were significantly increased in SP-SHR, shown also by peak current frequency distributions. There was a significant shift to the left of both activation (7 mV) and inactivation (15 mV) current-voltage (I-V) plots. SP-SHR IBa recovery from inactivation was significantly slower (103 versus 61 ms) than in N/nih VMC. The increases in SP-SHR IBa amplitude under maximized conditions correlated with increases in blood pressure. Together with earlier observations of increased vascular muscle Ca2+ current density coexistent with blood pressure elevation in Kyoto-Wistar SHR, these data provide evidence for altered function of Ca2+ channels as a fundamental component of hypertension. Since the Ca2+ channel alterations exist in venous VMCs of newborn SP-SHR rats (in a low pressure blood vessel and at a time when increased Ca2+ current density could not be an effect of increased blood pressure), our results add to the growing evidence of Ca2+ channel abnormalities as a cause of genetic hypertension.

Protein kinase C mechanism enhances vascular muscle relaxation by the Ca2+ antagonist, Ro 40-5967.

Actions of the novel Ca2+ antagonist, Ro 40-5967, which displays unusual efficacy against endothelin (ET)-induced contractions, were studied in isolated vascular muscle cells (VMCs) using the fluorescent protein kinase C (PKC) indicator, BODIPY 12 alpha-phorbol ester 13 beta-acetate (PBA-BODIPY). High-sensitivity (photon-counting) digital-imaging microscopy quantified PKC distribution within VMCs and showed translocation from the cytosol to the cell surface membrane on stimulation with ET. ET (1 nM) stimulated translocation of PBA-BODIPY fluorescence that peaked at 4 min, increasing from 19 +/- 2% to 29 +/- 2% surface membrane (edge) distribution (n=44, p<0.05). Increases in membrane-associated PKC due to translocation began within 2 min and persisted for about 10 min, after which a gradual decline to control levels occurred. Upon exposure to Ro 40-5967 (10 microM), there was an inhibition of fluorescence intensity throughout the cell. Average fluorescence intensity decreased to 84 +/- 4% (n=20, p<0.05) of that in prestimulus controls. Cell/membrane was also reduced to below unstimulated control levels. Amlodipine failed to decrease PKC fluorescence intensity or translocation to the surface membrane. These data suggest that there is an important direct PKC inhibitory action of Ro 40-5967 that would at least partially explain relaxation of ET-induced contractions.

Ca2+ channel actions of the non-dihydropyridine Ca2+ channel antagonist Ro 40-5967 in vascular muscle cells cultured from dog coronary and saphenous arteries

SummaryWe studied the membrane effects of (1S,2S)-2-(2-[[3-2(benzimidazolyl) propyllmethylamino]ethyl)-6-fluoro-1,2,3,4-tetrahydro-l-isopropyl-2-naphthyl-methoxy-acetate dihydrochloride, Ro 40-5967, a new non-dihydropyridine (DHP) Ca2+ channel antagonist, on dog coronary and saphenous arterial vascular muscle cells using the whole-cell patch-clamp method. Long-lasting (L-type) inward currents in 20 mM Ba2+ were measured over a range of test potentials (300 ms) from −50 mV to + 90 mV from a holding potential of −80 mV in the presence of 1 μM Bay k8644 (a DHP Ca2+ agonist). Ro 40-5967 caused a concentration-dependent suppression of Ca2+ channel currents in muscle cells from both arteries, with greater potency on coronary than saphenous arterial cells. The concentration of Ro 40-5967 which inhibited the magnitude of peak inward currents by 50% (IC50) was estimated to be 1 μM (n = 5) in muscle cells from coronary artery and 10 μM (n = 4) in saphenous artery. Ro 40-5967 (1 μM) decreased the amplitude of the activation current-voltage relationship for coronary L-type Ca2+ channel currents over a wider range of membrane potentials than verapamil, diltiazem, or nifedipine. In contrast, block of Ca2+ channel currents in saphenous artery cells by 1 μM Ro 40-5967 was only observed at command potentials positive to 0 mV. Ro 40-5967 (1 μM) significantly shifted the voltage-inactivation curve downward by 40% in coronary (n = 4), but only by 18% in saphenous arterial muscle cells (n = 3). The non-parallel shift of the coronary artery inactivation curve suggests that pronounced resting channel block is a notable feature of Ro 40-5967. The marked inhibition of Ba2+ current by 1 μM Ro 40-5967 in the inactivation protocol in coronary arterial muscle cells was found over the entire range of membrane holding potentials tested, while inhibition in the saphenous artery inactivation curve occurred only from holding potentials more positive than −40 mV. Therefore, Ro 40-5967 is unique: 1) in acting over a wider range of voltages, on both instantaneous and resting Ca2+ currents, than other Ca2+ antagonists; 2) in producing more significant resting state block; and 3) in acting with selectivity for coronary over saphenous arteries.

Reactivity-based coronary vasospasm independent of atherosclerosis in rhesus monkeys

OBJECTIVES We studied the hypothesis that in the absence of vascular pathology, coronary artery vasospasm occurs as a result of local regions of vascular muscle hyperreactivity. We aimed to explore the basis for a functional etiology of those vasospasms not explained on a structural basis. BACKGROUND Ovariectomized rhesus monkeys (Macaca mulatta) without injury or significant vascular disease were stimulated with platelet release products, and angiograms were compared with those from vasospasms induced in human patients. METHODS We used intracoronary (IC) injections of serotonin, thromboxane A2 (U46619), endothelin 1 or angiotensin II in concentrations 3 to 10 times that which reduced coronary artery diameter by 50%. RESULTS Although no agent alone caused vasospasm, the combination of pathophysiologic concentrations of serotonin and the stable thromboxane A2 mimetic, U46619, injected through an IC catheter, synergistically caused coronary vasospasm on the second or third challenge in five of seven monkeys. These drug-induced vasospasms were similar to vasospasms induced by mechanical injury followed by serotonin, and to those stimulated in human IC diagnostic tests, as judged by onset, appearance, kinetics and vasodilator reversal. CONCLUSIONS These studies in ovariectomized monkeys revealed that coronary vasospasm can be stimulated without preexisting vascular pathology, endothelial denudation or injury. Reproducible vasospasm of primate coronary arteries in response to these two endogenous pathophysiologic vasoconstrictors, which are thought to be precipitating stimuli in the etiology of vasospasm, suggests that structure-independent epicardial vasospasm can be an important element in serious cardiac ischemic events, particularly the focal, persistent vasospasms that occur without plaques or injury.

Role of T Channels in Cardiovascular Function

Although two types of Ca2+ channels are found to occur in the cardiovascular system, very little is known about one of them, primarily because a pharmacological blocking agent has been lacking. The enigmatic transient (T)-type Ca2+ channel has finally been recognized by a selective Ca2+ antagonist. The novel tetralol Ca2+ antagonist, mibefradil, is a selective T-type Ca2+ channel blocker that produces effective vasodilatation with additional inhibitory actions on blood vessel wall and left ventricular thickening. The availability of a blocking agent has begun to reveal the significance of T-type Ca2+ channel signals. Selective T-type Ca2+ channel blockade characteristics include vascular selectivity, freedom from negative cardiac inotropism, consistent and predictable reduction in heart rate, reduction in subendothelial proliferation, and increased survival of severely hypertensive and heart failure animal models. Mibefradil increases coronary blood flow without increasing myocardial oxygen consumption, and by decreasing heart rate and thus time spent in diastole, improves subendocardial perfusion. Improved perfusion of the myocardial wall and lowered heart rate appear to normalize the underlying pathophysiological factors, improve heart failure, and provide long-term protection. Thus, T-type Ca2+ channel blockade offers significant new cardiovascular protective benefits, even in the presence of critical pathophysiological elements (i.e. increased heart rate and neurohumors in the presence of decreased ejection fraction and contractility) found in heart failure.

GLYBURIDE ACTIONS ON THE DIHYDROPYRIDINE-SENSITIVE CA2+ CHANNEL IN RAT VASCULAR MUSCLE

The effects of glyburide, a purportedly selective ATP-sensitive K+ channel antagonist, were studied on dihydropyridine (DHP)-sensitive (L-type) Ca2+ channel currents in rat aortic muscle cells. Whole-cell voltage-clamp Ba2+ currents (IBa) were recorded at a series of test potentials (VT) from -30 to +60 mV during 300-ms voltage steps from a holding potential of -80 mV. Bay k8644 (1 microM) increased peak divalent cation currents from 47.2 +/- 15.1 to 102.6 +/- 13.4 pA, and the current-voltage relationship curve was shifted 10 mV to the left (n = 5). The combination of 10 microM glyburide with 1 microM Bay k8644 further increased Bay k8644-enhanced IBa in each cell (average of 223.7 +/- 26.4 pA, n = 5), and caused a further 10 mV hyperpolarizing (leftward) shift of the activation curve. The kinetics of IBa were also changed (more rapid inactivation) by glyburide. These stimulatory actions of glyburide were reversed on washout. In contrast to this apparent synergism with Bay k8644, 10 microM glyburide alone inhibited (rather than potentiated) IBa by about 20% at VT of 0, +10, and +30 mV. Increasing glyburide concentration to 30 microM further inhibited the IBa to about 40-50% of controls. With the pure agonist isomer, 0.5 microM Bay R5417, at theoretically the same concentration of the minus enantiomer as is present in Bay k8644, IBa increased from 137 +/- 18.3 pA to 354.2 +/- 12.4 pA (n = 4).(ABSTRACT TRUNCATED AT 250 WORDS)

Vascular muscle calcium channel modulation in hypertension

Indications of membrane alterations in vascular muscle cells of spontaneously hypertensive rats (SHR), compared to their Kyoto-Wistar nomotensive controls (WKY), have led to further investigation of calcium channels. Previous work from this laboratory had shown the increased probability for opening of the longer-lasting (L-type) calcium channels in SHR, suggesting differences in number or modulation. These experiments have been carried out on the azygos vein of neonatal rats because that preparation has been characterized electro-physiologically, pharmacologically, and by contractile parameters. Divalent (inward) ion currents through the L-type calcium channels are more readily carried by barium than by calcium, a characteristic that is not true for the transient (T) channels. Because there is an increased ratio of L to T calcium channels in SHR, the substitution of barium for calcium is more apparent for inward current amplitude in SHR than in WKY. This increase in the sustained L-type calcium currents, appearing without increased blood pressure on the venous side in newborn animals, is suggestive of a genetic membrane alteration that could contribute to vascular muscle membrane changes important in the development of increased blood pressure. Description and differentiation of the ribbon shaped vascular muscle cells from cardiac muscle cells, and the potential for confusion of the two in older animals, was addressed. The predominance of T-type calcium currents in these azygos vein cells, which is likely to correlate with the predominance of rapid spontaneous contractions, offers a compelling reason for selection of azygos veins in Ca2+ channel comparisons to establish etiologic factors at the cell level in hypertension.