Satish Batra

Estrogen receptors in the human female lower urinary tract

Cytosolic and nuclear fractions prepared from the urethra, urinary bladder, and trigonum of the bladder obtained at urethrocystectomy from four female patients were analyzed for the presence of estrogen receptors. High-affinity estradiol receptors (KD 0.7 x 10(-9)M) could be detected in both cytosolic and nuclear fractions of the urethra from all four patients. Estradiol receptors could be detected in only the nuclear fractions of the urinary bladder in two of the four preparations. In the trigonum, cytosolic and nuclear receptors could be measured in one and three preparations, respectively. The receptor concentrations in both trigonum and the bladder were lower than those in the urethra. By providing experimental evidence for the presence of estradiol receptors in the lower uninary tract, the present data advance the case for estradiol therapy in incontinent patients.

Female urethra: a target for estrogen action.

It is not uncommon to use estrogen therapy in patients with urinary stress incontinence. The possibility of a selective action of estrogen in the lower urinary tract was examined. Wet weight of the uterus, vagina and urethra increased significantly, and that of the urinary bladder insignificantly after estradiol treatment of ovariectomized rabbits. When ovariectomized rabbits were injected i.v. with 3H-estradiol, the tritium concentration, determined after 1 hour, was 3 to 4 times higher in urethra, urinary bladder and vagina than that in the muscle. High affinity estradiol receptors (KD approximately 1 X 10(-9) M) could be demonstrated in both the cytoplasmic and nuclear fractions prepared from the female rabbit urethra and bladder. The concentrations of estradiol receptors in the urethra and bladder were about 10 and 20 times lower respectively than those in the uterus. The present evidence for estradiol receptors in the lower urinary tract supports the case for estradiol therapy in urinary incontinence.

Progesterone receptors in the female lower urinary tract.

When female estrogenized rabbits were injected i.v. with 3H-progesterone, the tritium concentration determined after one hour was about two to three times higher in urethra, urinary bladder and vagina than in the heart. High affinity progesterone receptors (KD = 1-2 nM) could be demonstrated in both cytoplasmic and nuclear fractions prepared from estrogenized rabbit urethra, bladder and vagina. The cytosolic receptor concentration in both urethra and bladder was about half of that in the vagina. The concentration of nuclear receptors in urethra was not significantly different from that in the vagina, but in the bladder the concentration was only about one fourth of that in the vagina or urethra. The mean KD of cytosolic receptors from bladder was significantly higher than the corresponding values in urethra and vagina. Progesterone binding sites in the bladder had a broader hormonal specificity than those in the urethra or vagina. The present demonstration of specific progesterone receptors in the female urethra might provide a possible link between estrogen progesterone interaction and the appearance of urinary incontinence during pregnancy in women.

The effects of drugs on calcium uptake and calcium release by mitochondria and sarcoplasmic reticulum of frog skeletal muscle

Abstract The effects of quinidine, chlorpromazine and caffeine on Ca uptake and Ca release by mitochondria and fragmented sarcoplasmic reticulum (FSR) of frog muscle were studied. Quinidine (1–2 mM) released considerable Ca from preloaded mitochondria but had little effect on preloaded FSR. The uptake of Ca both by mitochondria and FSR was inhibited by higher concentrations (2 or 1 mM) of quinidine but the inhibition of mitochondrial Ca uptake was much greater. With lower concentration (0.4 mM), there was no significant effect on Ca uptake by FSR, but a 48 per cent inhibition of mitochondrial Ca uptake was observed. Chlorpromazine (0.01–0.1 mM) inhibited Ca uptake by both mitochondria and FSR, but the inhibition in the case of FSR was weaker than mitochondria. Only the highest concentration (0.1 mM) of chlorpromazine caused a release of Ca from mitochondria or FSR. Caffeine (2–10 mM) inhibited Ca uptake both by mitochondria and FSR and again the inhibition of Ca uptake by mitochondria was greater than of FSR. Caffeine (10 mM) in contrast to quinidine released Ca from FSR and not from mitochondria. Ca releasing effect of both caffeine and quinidine increased when the ratio of the drug and FSR protein increased. The Ca releasing concentrations of these drugs were comparable to those reported to elicit contractures of living muscle. The lower concentrations which inhibited Ca uptake were comparable to those which potentiate twitch.

Effect of estrogen and progesterone treatment on calcium uptake by the myometrium and smooth muscle of the lower urinary tract

In order to elucidate the mechanisms by which estrogen treatment increases uterine contractility, the effect of estrogen on Ca uptake was measured in the isolated rabbit uterus as well as in the urethra and urinary bladder. Estrogen treatment more than doubled the amount of cellular Ca uptake in the uterus during both rest and potassium-induced depolarization. Combined estrogen and progesterone treatment had a similar effect to estrogen alone. In both urinary bladder and the urethra, treatment with estrogen alone, or estrogen combined with progesterone had no effect on Ca uptake. Cellular Ca uptake was higher in uteri of rabbits subjected to progesterone withdrawal than in those that were under progesterone dominance. It is concluded that estrogen-induced increase in Ca entry into the uterine smooth muscle cells is a likely mechanism underlying increased uterine contractility. Since this effect of estrogen was not antagonized by progesterone, it may not strictly rely on the genomic action of estrogen. The increase in cellular calcium following progesterone withdrawal could be one of the mechanisms for the evolution of myometrial activity at parturition.

Acute Chlamydia pneumoniae infection causes coronary endothelial dysfunction in pigs.

BACKGROUND Coronary endothelial dysfunction contributes to the pathogenesis of acute coronary syndromes (ACSs). Acute Chlamydia pneumoniae infection has been epidemiologically associated with ACS. In this study, we investigated whether acute C. pneumoniae infection could alter the endothelial vasomotor function of porcine coronary vessels. METHODS AND RESULTS Twenty pigs, 7-9 kg in weight, were inoculated intratracheally with C. pneumoniae (n=12) or saline (n=8), and investigated at 3 days (five infected/four non-infected) and 2 weeks (5+2 infected/four non-infected) after inoculation. The endothelium-dependent reactivity of coronary microcirculation was assessed at both time points by measuring peak coronary flow velocity (CFV) in response to bradykinin, before and after infusions with glutathione, an antioxidant, and L-arginine, a substrate for nitric oxide synthase (NOS). CFV after bradykinin was significantly decreased in infected animals at both time points. At 2 weeks, both glutathione and L-arginine significantly improved CFV after bradykinin. CFV after sodium nitroprusside (SNP) was similar in both groups. At 3 days, the relaxation responses of bradykinin-induced pre-contracted left anterior descending (LAD) coronary rings to bradykinin were significantly less in infected animals. N(G)-nitro-L-arginine-methyl-ester, an NOS inhibitor, had significantly greater inhibitory effect on bradykinin-induced relaxation in infected animals. Plasma nitrate-nitrite and fibrinogen, and NOS activity from LAD coronary samples were significantly increased in infected animals. CONCLUSION Acute C. pneumoniae infection causes endothelial dysfunction of both resistance and epicardial coronary vessels, and favours a pro-coagulant status. These effects could in part account for the epidemiologically suggested association between acute infection and ACS.

Characterization of nitric oxide synthase activity in rabbit uterus and vagina: Downregulation by estrogen

Nitric oxide synthase (NOS) was determined in the soluble (cytosolic) and particulate fractions of rabbit uterus, vagina and cerebellum and the influence of estrogen treatment on NOS activity was studied. NOS in both the cytosolic and particulate fractions was highly calcium dependent. The activity in cytosolic fraction was nearly 4-fold higher than the particulate fractions from all three organs. The concentration of NOS was highest in cerebellum followed by vagina and uterus. Vaginal NOS activity was 3-4-fold higher than the uterine NOS. After a continuous treatment of rabbits for one week with estrogen, cytosolic NOS was reduced by nearly 7 and 4-fold in the uterus and vagina, respectively, whereas there was no significant change in the particulate NOS. Estrogen treatment caused no change in cytosolic or particulate NOS from the cerebellum. Downregulation of cytosolic NOS by estrogen in the estrogen target tissues like uterus and vagina and absence of effect in the cerebellum strongly suggests a physiological significance.

Nitric Oxide Synthase in the Rabbit Uterus and Vagina: Hormonal Regulation and Functional Significance

Abstract The effects of estrogen (E2), progesterone (P4), and E2 and P4 (E2+P4) on uterine, vaginal, and cerebellar nitric oxide synthase (NOS) were examined. Additionally, experiments were done to investigate whether NOS-containing nerves were present in the uterus and vagina and the extent to which vaginal smooth muscle response was dependent on nitric oxide (NO). Cytosolic NOS was determined by the formation of [14C]citrulline from [14C]arginine, and NOS localization was visualized by immunohistochemistry. Vaginal smooth muscle relaxation was induced by electrical field stimulations (EFS). NOS activity in the uterus was markedly down-regulated in all hormone-treated groups. Vaginal NOS activity was nearly 4-fold higher than the uterine NOS activity and was considerably reduced by E2 or E2+P4 treatment. In contrast to findings in the uterus, P4 treatment up-regulated vaginal NOS. Hormone treatment had no significant effect on cerebellar NOS. NOS-containing nerves could be demonstrated in the uterus and vagina by immunohistochemistry. Vaginal smooth muscle responded with relaxation after EFS, which was inhibited by NG-nitro-l-arginine. A relatively high vaginal NOS, a down-regulation by E2, an up-regulation by P4, and NO-dependent response of vaginal smooth muscle suggest a tissue-specific physiological role.

Effect of ovarian steroids and diethylstilbestrol on the contractile responses of the human myometrium and intramyometrial arteries

Estriol, estradiol, progesterone and diethylstilbestrol in the concentration range of 0.2-40 microM inhibited the spontaneous contractions of the myometrium in a dose-dependent manner but the differences in IC50 values obtained with different hormones were not statistically significant. All these hormones caused a concentration-dependent inhibition of the K(+)-induced contraction. The IC50 values were lowest for diethylstilbestrol and highest for estriol. Vasopressin at concentrations of 1.5 x 10(-6) - 1.8 x 10(-3) U/ml stimulated myometrial contractions. These responses were also inhibited by ovarian steroids and diethylstilbestrol. The IC50 values for estriol and progesterone were significantly higher than for estradiol or diethylstilbestrol. The values for estriol and progesterone did not differ significantly. In the uterine arteries, which lacked spontaneous activity, ovarian steroids and diethylstilbestrol inhibited contractions induced by K+ depolarization. As with myometrium, the lowest effect was observed with estriol and the highest with diethylstilbestrol. A dose-dependent inhibition by all four hormones (0.2-40 microM) of vasopressin-induced contractile responses of the uterine arteries was observed. With the lowest concentration of progesterone, however, the arterial response to vasopressin was enhanced. The increases by progesterone (0.02 and 0.2 microM) of responses induced by vasopressin were statistically significant (P < 0.05). The present data strongly suggest that, in human myometrium and uterine arteries, ovarian steroids and diethylstilbestrol cause a more pronounced inhibition of receptor-mediated than of voltage-dependent Ca2+ channels. The increase by a very low (physiological) concentration of progesterone of vasopressin-induced responses in both myometrium and arteries may be of significance in the pathophysiology of dysmenorrhea.

Effect of some estrogens and progesterone on calcium uptake and calcium release by myometrial mitochondria.

Abstract Uptake of Ca by human myometrial mitochondria was inhibited by as low as 4 μM of diethyl stilbestrol (DES) in the medium, and 20 μM inhibited Ca uptake almost completely. Ethinyl estradiol and estradiol-17β also inhibited Ca uptake but the inhibition was smaller than DES and in the order DES > ethinyl estradiol > estradiol-17β. None of the agents had an effect on the mitochondrial ATPase in concentrations that inhibited Ca uptake. Progesterone did not affect either the Ca uptake or the ATPase activity. DES was also able to release Ca stored by mitochondria in concentrations that inhibited Ca uptake. Addition of supernatant fraction in the medium afforded protection from DES inhibition of Ca uptake. These findings together with those reported previously lend support to the argument for a role of mitochondria in contraction and relaxation of human myometrium.