Estrogen enhances basal nitric oxide release in the forearm vasculature in perimenopausal women.

The mechanisms of estrogen-induced cardiovascular protection are incompletely understood. Acute estrogen administration enhances acetylcholine-induced vasorelaxation, suggesting that endothelium-dependent factors may be important. The effect of long-term estrogen supplementation on endothelial function has not been well defined. In this double-blind, randomized study, we examined endothelial function in forearm resistance arteries in 11 perimenopausal women before and after 8 weeks of estrogen supplementation (estradiol valerate, 2 mg daily, n = 6) or placebo (n = 5). Forearm blood flow was measured by venous-occlusion plethysmography, and vasoactive agents were infused through a brachial artery cannula in doses that did not influence blood pressure or heart rate. Estrogen supplementation significantly reduced systolic and diastolic pressures but had no effect on plasma lipoproteins. Estrogen did not alter the vasodilator responses to acetylcholine at doses of 9.25, 18.5, and 37 micrograms/min (rise in forearm blood flow before estrogen: 263 +/- 72%, 288 +/- 66%, and 383 +/- 84%, respectively; after estrogen: 205 +/- 34%, 260 +/- 44%, and 359 +/- 54%, P > .05.). Vasodilator responses to the endothelium-independent agent sodium nitroprusside (1.6 micrograms/min) were also unchanged after estrogen supplementation. However, estrogen enhanced vasoconstrictor responses to the nitric oxide synthase inhibitor NG-mono-methyl-L-arginine at doses of 1, 2, and 4 mumol/min (fall in fore-arm blood flow before estrogen: 13 +/- 9%, 20 +/- 7%, and 26 +/- 8%, respectively; after estrogen: 18 +/- 9%, 36 +/- 7%, and 47 +/- 7%, P = .04). Responses to vasoactive agents were unchanged after administration of placebo. Thus, in perimenopausal women, estrogen supplementation reduces blood pressure and enhances basal but not acetylcholine-induced nitric oxide release in fore-arm resistance arteries.

Localization of 11beta-hydroxysteroid dehydrogenase type 2 in rat tissues: in situ studies.

In the rat, the enzyme 11beta-hydroxysteroid dehydrogenase 2 (11betaHSD2) converts the glucocorticoid corticosterone into receptor-inactive 11-dehydrocorticosterone, thereby allowing preferential access of aldosterone to mineralocorticoid receptors (MR). The present study examines the distribution of this enzyme by in situ hybridization, using a homologous complementary RNA probe for 11betaHSD2. 11betaHSD2 messenger RNA was detected in classic epithelial aldosterone target tissues (kidney, salivary glands, and colon), the female reproductive system (ovary, oviduct, uterus, and placenta), and the adrenals; levels in heart, testis, and liver were below the limits of detection. We interpret the finding of 11betaHSD2 expression in both classical MR-containing aldosterone target tissues and a variety of other tissue as evidence that in the rat, the enzyme may play physiological roles in addition to that of excluding glucocorticoids from epithelial MR.

Hormone-nuclear receptor interactions in health and disease. Mineralocorticoid resistance.

Mineralocorticoid resistance, or pseudohypoaldosteronism (PHA), is a rare cause of salt wasting in young children. It may be inherited as an autosomal dominant or recessive trait, it may occur sporadically or, rarely, it may develop secondary to other conditions. It is characterized by episodes of dehydration and hyponatraemia in the face of high aldosterone levels. In most cases, after a short period of salt supplementation no further ill effects are experienced. The condition is of great interest because it provides insights into both the mechanisms by which salt and water balance are controlled and the actions of aldosterone. This article reviews the normal physiology of aldosterone, with particular reference to its biosynthesis and its actions in specific target tissues. Current knowledge regarding the molecular mechanisms involved in aldosterone action is discussed in some detail. The clinical features of PHA are reviewed and diagnostic issues and clinical management considered. Finally, current views regarding the pathophysiology of the condition are presented. Here, considerable uncertainty remains. Whilst in many cases of PHA there is greatly reduced binding of aldosterone to its receptor, the underlying abnormality is yet to be identified; in particular, in spite of strong reasons for suspecting a defect or defects in the mineralocorticoid receptor, there is so far no direct evidence to support this hypothesis. The article concludes with a discussion of other possible explanations for the underlying abnormality in PHA.

Evidence that the central noradrenergic and adrenergic pathways activate the hypothalamic-pituitary-adrenal axis in the sheep.

These studies were undertaken to test the hypothesis that stimulation of the central noradrenergic and adrenergic pathways activates the hypothalamic-pituitary-adrenal axis in vivo in the conscious sheep. Blood samples were taken at 10-min intervals over 4 h to establish the baseline state, and then each animal received an intracerebroventricular (icv) injection of NaCl (control animals) or catecholamine [norepinephrine (NE) or epinephrine (EPI)]. A more frequent rate of venous sampling was used for the 30-min period after the icv injection, after which time the 10-min rate of blood sampling was continued for another 3.5 h. NaCl (n = 4) caused no change in pituitary-adrenal secretion. In contrast, 10 micrograms NE (n = 4) caused acute 1.9- and 3.2-fold increases in mean plasma ACTH and cortisol levels over the 1 h period post injection, and 1.6- and 2.3-fold increments in their concentrations over the 4 h postinjection period. Although 10 micrograms EPI (n = 4) did not elevate mean plasma ACTH, it produced significant 1.7- and 1.5-fold increases in plasma cortisol during the 1- and 4-h periods post injection. However, when 100 micrograms EPI was injected (n = 4), acute 9.5- and 5.5-fold increases in plasma ACTH and cortisol were seen over the 1 h period post injection, and 6.1- and 4.2-fold increments in their plasma concentration were noted during the entire post-injection period. To determine the predominant site of action of the catecholamines, we also examined the ability of NE and EPI to release ACTH from cultured ovine anterior pituitary cells. NE and EPI (10(-9)-10(-6) M) stimulated the release of ACTH in a dose-dependent manner, but with maximal increments only 1.5-fold greater than the basal secretion. NE and EPI also increased the maximal ACTH response to CRF, but did not alter the maximal ACTH release induced by arginine vasopressin.(ABSTRACT TRUNCATED AT 400 WORDS)

Characterization of 11 beta-hydroxysteroid dehydrogenase gene expression: identification of multiple unique forms of messenger ribonucleic acid in the rat kidney.

The enzyme 11 beta-hydroxysteroid dehydrogenase (11-HSD) appears to be involved in mediating aldosterone specificity of otherwise nonselective type I receptors in mineralocorticoid target tissues. In the present study gene expression of 11-HSD was characterized in various tissues of the rat by use of a complementary DNA probe coding for the rat liver 11-HSD. In the liver, lung, testis, colon, heart, hippocampus, and kidney papilla a single message was observed of length approximately 1700 nucleotides (nt). In the kidney cortex/medulla, however, messenger RNA (mRNA) species were observed at 1900 nt, 1600 nt and 1500 nt, and deadenylation studies showed that the renal 1900 nt species was heterogeneous. Northern blot analysis of 11-HSD mRNA showed low levels of expression in the kidney of the neonate and much higher levels in liver and lung with expression increasing markedly in all three tissues over development. In mature rats, a low salt diet significantly elevated 11-HSD mRNA in the liver but not in other tissues. We interpret these data as evidence for the existence of a family of 11-HSD genes, and consistent with the possibility that the hepatic species may modulate occupancy of type II (classical) glucocorticoid rather than type I receptors.

Studies of the regulation of the hypothalamic-pituitary-adrenal axis in sheep with hypothalamic-pituitary disconnection. II. Evidence for in vivo ultradian hypersecretion of proopiomelanocortin peptides by the isolated anterior and intermediate pituitary.

Studies were performed to determine whether the isolated ovine anterior and intermediate pituitary might rhythmically secrete three POMC peptides, ACTH, ir-beta-endorphin (ir-beta-EP), and ir-alpha-melanocyte stimulating hormone (ir-alpha-MSH) in vivo. When blood was taken at 10-min intervals from four ewes with hypothalamo-pituitary-disconnection (HPD), a distinct POMC-peptide and cortisol ultradian rhythm was noted. A comparison of the four HPD ewes with five nonstressed hypothalamopituitary-intact (HPI) ewes revealed that the mean plasma levels of the three POMC-peptides and cortisol were increased, the mean ACTH and ir-alpha-MSH pulse amplitudes were increased, and the mean ir-beta-EP and ir-alpha-MSH interpulse intervals were decreased. When four HPI ewes were subjected to a mild stress, plasma POMC-peptide and cortisol levels increased significantly when compared with the five unstressed HPI animals. In addition, the ACTH and cortisol pulse amplitudes increased and the ir-beta-EP and ir-alpha-MSH interpulse intervals decreased. Although plasma ACTH levels in the stressed HPI and HPD ewes were comparable, mean plasma cortisol levels were 2-fold greater in the stressed HPI animals. To determine whether the ACTH hypersecretion in the HPD ewe might reflect a net reduction in hypothalamic inhibitory influence over ACTH secretion, we examined the effects of dopamine (DA), somatostatin (SS-14), and rat atrial natriuretic peptide [rANF(1-28)] on the secretion of ACTH from cultured ovine anterior pituitary cells. DA and SS-14 did not exert a discernible effect on basal, CRF-, or arginine vasopressin (AVP)-stimulated ACTH secretion. Although basal ACTH secretion was unaffected by rANF(1-28) (10(-12)-10(-8) M), a significant inhibition of CRF- and AVP-stimulated ACTH release was observed.(ABSTRACT TRUNCATED AT 400 WORDS)

Regulation of follicle-stimulating hormone beta and common alpha-subunit messenger ribonucleic acid by gonadotropin-releasing hormone and estrogen in the sheep pituitary.

The effect of gonadotropin-releasing hormone (GnRH) and/or estradiol (E2) on pituitary messenger ribonucleic acid (mRNA) levels of luteinizing hormone beta (LH beta), follicle-stimulating hormone beta (FSH beta) and the common alpha-subunit were determined in anterior pituitary glands from ovariectomized (OVX) ewes. Hypothalamo-pituitary disconnected (HPD) ewes receiving appropriate hormonal treatment were used to assess the relative roles of GnRH and E2 in directly regulating FSH beta and alpha-subunit mRNA levels. Levels of LH beta mRNA were increased in OVX animals compared with intact controls, and E2 treatment of OVX animals significantly reduced mRNA levels of LH beta and FSH beta. HPD substantially reduced FSH beta and alpha-subunit mRNA levels. Treatment of OVX/HPD animals with pulses of GnRH (250 ng/2 h) for 1 week restored FSH beta and alpha-subunit mRNA to OVX levels. Combined GnRH and E2 treatment significantly lowered FSH beta mRNA levels, but resulted in a rise in alpha-subunit mRNA levels. Treatment of OVX/HPD ewes with E2 alone had no effect on FSH beta and alpha-subunit mRNA levels. These findings indicate that E2 acts directly on the pituitary to negatively regulate FSH beta mRNA levels, and to positively regulate alpha-subunit mRNA levels in the presence of GnRH.

Glucocorticoid regulation of proopiomelanocortin gene expression in the pituitary gland of hypothalamopituitary intact and hypothalamopituitary disconnected sheep.

Proopiomelanocortin (POMC) gene expression in the anterior pituitary (AP) gland has previously been shown to be positively regulated by CRF and AVP and negatively regulated by glucocorticoids. In the neurointermediate lobe (NIL) of the pituitary, however, POMC gene expression is under tonic inhibitory dopaminergic control. In the present study we have used hypothalamopituitary intact (HPI), ovariectomized (OVX), and OVX/hypothalamopituitary disconnected (OVX/HPD) ewes to examine direct (i.e. nonhypothalamic) effects of glucocorticoids on POMC gene expression in both the AP and the NIL. There was no difference between POMC mRNA levels in intact and OVX sheep. In intact animals treated with dexamethasone, AP POMC mRNA levels were half those of controls. POMC mRNA levels were increased 3-fold in OVX/HPD sheep, compared with OVX, and lowered by dexamethasone to half OVX/HPD levels. In the NIL, hypothalamopituitary disconnection resulted in slightly higher mean POMC mRNA levels than in intact animals but the large intragroup variation did not allow a significant change. Dexamethasone administration had no effect on NIL levels of POMC mRNA in intact or OVX/HPD sheep.

Immunolocalization of renal mineralocorticoid receptors with an antiserum against a peptide deduced from the complementary deoxyribonucleic acid sequence.

We have synthesized three peptides corresponding to putative antigenic regions in the immunogenic domain, hinge region, and carboxy-terminus of the protein. A rabbit immunized with a peptide derived from the hinge region of the receptor produced an antiserum which showed 50% displacement with 20 pg peptide at a final serum dilution of 1:35,000. When the antiserum was immunopurified and applied to sections of intact rat and human kidney it stained cells lining segments corresponding to distal tubule, connecting piece, and initial cortical collecting duct, consistent with the known sites of mineralocorticoid action. In both human (formaldehyde-fixed) and rat (Bouin's solution) there was ample evidence for both nuclear and cytoplasmic staining. The thymus, in which previously we have found [3H]aldosterone binding to be below detection limits, showed little or no staining. Western blot analyses demonstrated that the polyclonal antibody recognized an epitope of the expected molecular size. The availability of antibodies to the mineralocorticoid receptor should, thus, facilitate investigation of the steroid specificity-conferring mechanism which allows mineralocorticoids, but not glucocorticoids, access to the nonselective receptor in the kidney.

Studies of the secretion of corticotropin-releasing factor and arginine vasopressin into the hypophysial-portal circulation of the conscious sheep. I. Effect of an audiovisual stimulus and insulin-induced hypoglycemia.

Studies were undertaken to characterize the secretion of corticotropin-releasing factor (CRF) and arginine vasopressin (AVP) into the hypophysial-portal circulation of the conscious sheep. In addition, we examined the temporal relationship between the secretion of these two hypothalamic peptides and the secretion of three pro-opiomelanocortin peptides--adrenocorticotropic hormone (ACTH), ir-beta-endorphin, and ir-alpha-melanocyte-stimulating hormone--and cortisol and determined the effects of an audiovisual emotional stimulus and insulin-induced hypoglycemia on the entire hypothalamic-pituitary-adrenal axis. In the basal state, the secretion of CRF, AVP, the three pro-opiomelanocortin peptides, and cortisol was pulsatile in nature, and three CRF and AVP pulse patterns were observed: a concordant increase in CRF and AVP, an isolated rise in CRF, and an isolated increase in AVP. In 4 of the 5 animals, a 3-min audiovisual stress (barking dog) rapidly increased the plasma levels of all the measured substances, although the magnitude and duration of the effect differed markedly between the animals. Insulin-induced hypoglycemia markedly increased AVP and, to a lesser extent, CRF concentrations in portal plasma and thereby altered the CRF:AVP molar ratio. Although pituitary-adrenal activation was closely correlated with the increased hypothalamic activity, a strict 1:1 concordance between CRF/AVP secretion and ACTH secretion was not seen. The anesthetic ketamine selectively increased portal AVP concentrations to levels which exceeded those attained during hypoglycemia and rapidly activated the pituitary-adrenal axis. We conclude the following: (1) CRF and AVP are secreted by the hypothalamus in a pulsatile fashion; (2) ACTH secretion can be stimulated by increases in either CRF or AVP; (3) the absence of a strict 1:1 concordance between hypothalamic CRF/AVP release and pituitary ACTH secretion during stress may be partly due to the release of additional hypothalamic ACTH secretagogues; (4) the ability of both audiovisual stimuli and insulin-induced hypoglycemia to augment CRF and AVP secretion indicates that the paraventricular hypothalamus may be activated by a variety of neural inputs, and (5) the marked alteration of the CRF:AVP molar ratio during stress suggests that AVP may be an important ACTH secretagogue in vivo in the sheep.

Gonadal steroids and anterior lobe dynorphin in the male rat.

Immunoreactive (ir)-dynorphin levels were measured, and the species characterized by high performance liquid chromatography (HPLC), in the pituitary and hypothalamus of intact and castrate male rats. On HPLC, ir-dynorphin co-eluted with authentic dynorphin A 1-8, dynorphin A 1-17 and dynorphin 1-32 in the hypothalamus and intermediate lobe; in two different reversed phase (RP)-HPLC systems, anterior lobe ir-dynorphin co-eluted uniquely with dynorphin 32 (4K dynorphin). Anterior lobe levels of total ir-dynorphin were significantly lowered 7 days after castration, while HPLC profiles in all tissues remained unchanged. The change in anterior pituitary ir-dynorphin levels was reversed in a dose-related manner by dihydrotestosterone (15-500 micrograms/100 g b. wt/day); estradiol benzoate (3 micrograms/100 g/day) was without effect. The changes on castration and androgen administration suggest that gonadal steroids play a role in the regulation of dynorphin, as well as gonadotrophins and prolactin, within the anterior pituitary gland.

Immunoreactive ACTH, beta-endorphin, and cortisol levels in plasma following spinal manipulative therapy.

This study examines the possibility of a humorally mediated analgesic response to spinal manipulative therapy by determination of plasma levels of beta-endorphin, adrenocorticotropic hormone (ACTH), and cortisol before and after intervention. Forty male subjects (20 symptomatic, 20 asymptomatic) were allocated into four equal groups. Two treatment groups were given spinal manipulative therapy, and two groups underwent a sham procedure. Blood samples were taken via indwelling butterfly needles pre- and postintervention in all four groups, and levels of immunoreactive ACTH, immunoreactive beta-endorphin, and cortisol determined by radioimmunoassay. No differences in ACTH or beta-endorphin were found between sham and treated groups, or between pre- and postintervention in any group; cortisol levels fell over the course of the study in all groups. The findings thus appear to exclude a humoral role for beta-endorphin in mediating the analgesic response to spinal manipulative therapy; in addition, they suggest that such therapy is not a stressor that activates the hypothalamo-pituitary-adrenal axis.

Studies of the regulation of the hypothalamic-pituitary-adrenal axis in sheep with hypothalamic-pituitary disconnection. I. Effect of an audiovisual stimulus and insulin-induced hypoglycemia.

These studies were undertaken to characterize the secretion of adrenocorticotropin (ACTH), immunoreactive (ir) beta-endorphin (ir-beta-EP) and ir alpha-melanocyte-stimulating hormone (ir-alpha-MSH) from the surgically isolated ovine pituitary in response to an audiovisual stress (barking dog, 3 min) and insulin hypoglycemia. The studies were performed in 4 ovariectomized, hypothalamo-pituitary-disconnected (HPD) and 4 sham-HPD ewes bearing indwelling jugular venous catheters. Basal concentrations of the three pro-opiomelanocortin (POMC) peptides and plasma cortisol were significantly increased in the HPD animals. When the control ewes were exposed to the audiovisual stimulus, plasma ACTH, ir-beta-EP and ir-alpha-MSH levels were increased 2.5-, 10-, and 5-fold 1 min after the stress; plasma cortisol attained maximal values at 5 min. In contrast, plasma levels of the three POMC peptides were not significantly increased in the HPD animals, although a rise in plasma cortisol occurred. The administration of regular insulin (5 units/kg i.v.) to control ewes caused plasma ACTH, ir-beta-EP, and ir-alpha-MSH levels to increase 17-, 22-, and 67-fold at 50 min; plasma cortisol values were maximal at 60 min. In contrast, the elevated basal levels of POMC peptides in the HPD animals were not significantly increased by the hypoglycemia, but a significant elevation of plasma cortisol was seen. We conclude that: (1) the increase in ACTH in intact animals after an audiovisual emotional stress and hypoglycemia, and the abolition of this increase by HPD, indicates that both stimuli, each acting through distinct neuroanatomical pathways, increase the net corticotropin-releasing activity of the hypothalamus; (2) the rise in plasma cortisol in HPD animals after stress suggests that peripheral humoral factors may release additional small amounts of ACTH from the anterior pituitary, and (3) the finding of increased basal ACTH levels after HPD suggests that POMC peptide synthesis and secretion by the anterior pituitary is tonically regulated by an inhibitory factor of hypothalamic origin.

Ontogeny of corticotropin-releasing factor and arginine vasopressein in the rat.

The ontogeny in the rat hypothalamus of arginine vasopressin (AVP) and corticotropin-releasing factor (CRF) was studied to determine whether these two peptides develop in parallel, given the synergistic effect of AVP with CRF on adrenocorticotropic hormone (ACTH) release. Hypothalami and extrahypothalamic cerebrum of 15-day fetal--40-day postpartum rats were extracted in acetic acid for radioimmunoassay. Both AVP- and CRF-like immunoreactivity was detected in whole brain extracts of 15-day fetuses. Hypothalamic CRF levels were low during fetal life, fluctuated around the time of birth, and then progressively rose to 63% of adult levels by day 21 postpartum. Extrahypothalamic levels of brain CRF paralleled hypothalamic levels. Hypothalamic AVP levels began to rise at fetal day 19 in a stepwise manner, with the greatest rise in levels occurring after day 14 postpartum. At day 21 postpartum hypothalamic levels were only 17% of adult. Extrahypothalamic AVP levels initially paralleled those in the hypothalamus but reached a plateau after day 4 post-partum, and did not rise again until after day 21. Hypothalamic CRF and AVP thus do not develop strictly in parallel; the adult ratios of these peptides (approximately 1:40) were never seen during development, perhaps of significance in the observed blunting of the hypothalamic-pituitary-adrenal response to stress in infant rats.

Immunoreactive dynorphin is regulated by estrogen in the rat anterior pituitary.

The pituitary and hypothalamic content of dynorphin was determined by radioimmunoassay and characterized by high-performance liquid chromatography (HPLC) in adult female Sprague-Dawley rats, intact and ovariectomized with and without estrogen treatment. Animals were given estradiol benzoate, or vehicle (oil) by six daily intramuscular injections. Anterior pituitary content of immunoreactive (ir)-dynorphin in ovariectomized rats was approximately twice that of intact animals, and consisted of a single HPLC peak co-eluting with dynorphin 32. Administration of estradiol benzoate (0.06-6 micrograms/day) caused a marked decrease of ir-dynorphin in the anterior lobe of castrate female rats, with a half-maximal effect at 0.2 microgram/day; levels were restored to those seen in intact animals with 6 micrograms estradiol benzoate per day, an effect which was not influenced by concomitant administration of progesterone (1 mg/day), or bromocriptine (100 micrograms/day). In the hypothalamus and neuro-intermediate lobe multiple peaks of immunoreactive dynorphin were seen, coeluting with dynorphin A 1-8, dynorphin A 1-17 and dynorphin 32. Neither castration nor estrogen treatment altered ir-dynorphin content in these tissues. These findings suggest that the ovary exerts a specific modulating influence on AP ir-dynorphin in the rat, and that in addition this inhibition appears to be mediated by ovarian estrogen.

Concomitant dopaminergic and glucocorticoid control of pituitary proopiomelanocortin messenger ribonucleic acid and beta-endorphin levels.

Synthesis and secretion of POMC-derived peptides appear to be differentially regulated in the anterior pituitary (AP) and neurointermediate lobe (NIL). In the AP, glucocorticoids inhibit, and CRF and arginine vasopressin stimulate, synthesis of POMC and release of immunoreactive (ir)-beta-endorphin (beta EP); in the NIL, synthesis and release of POMC and its derivatives are under tonic inhibitory dopaminergic control. There is, however, evidence for some overlap of these control mechanisms under certain circumstances. In the present study we have used specific RIA and Northern blot analysis to examine the effects of chronic treatment with dopaminergic agents and dexamethasone (DM) (both alone and in combination) on AP and NIL content of ir-beta EP and POMC messenger RNA (mRNA), and/or hypothalamic ir-arginine vasopressin and ir-CRF content. In the NIL, the dopamine agonist bromocriptine reduced and the antagonist haloperidol raised both POMC mRNA and ir-beta EP content. Long term DM treatment did not alter NIL ir-beta EP content in the intact rat, but increased levels of POMC mRNA. DM abolished the haloperidol-induced increase in NIL ir-beta EP content but further increased the haloperidol-induced rise in POMC mRNA. DM treatment lowered both ir-beta EP and POMC mRNA in the AP as well as lowering levels of hypothalamic ir-CRF. In DM-treated rats, haloperidol partially restored AP ir-beta EP and POMC mRNA to control untreated levels. These findings further support the proposition that both dopaminergic agents and glucocorticoids can modulate POMC mRNA levels and/or tissue content of ir-beta EP in both the NIL and AP of the rat. The effects of DM on the NIL, both alone or with haloperidol, suggest that glucocorticoids may have both direct and indirect effects on POMC gene expression in this tissue.

Isolation of opiate receptor ligands in coffee.

1. We have reported previously that instant coffee contains ligands for opiate receptors with characteristics similar to those of opiate antagonists. 2. A concentrate of receptor-active ligands from instant coffee was prepared by serial treatments involving Amberlite XAD-2, flash chromatography and gel permeation chromatography. 3. Examination of the final concentrate by GC-MS showed the presence of a number of isomeric (iso)feruloylquinic acid lactones. 4. It is suggested that the synthesis and biological testing of each quinide isomer will establish which is responsible for the opiate receptor activity of instant coffee.

Expression of the renal kallikrein gene in mineralocorticoid-treated and genetically hypertensive rats.

On the basis of both clinical observations and experimental studies it has been proposed that renal kallikrein is a mineralocorticoid regulated protein. In other studies, changes in renal kallikrein activity have been implicated in the genesis of, and/or response to, hypertension. Using a cloned complementary DNA (cDNA) to rat pancreatic kallikrein (pcXP39) for hybridization histochemistry, and both Northern and dot blot analysis, we studied expression of the kallikrein gene in steroid-treated control animals, and in three strains of genetically hypertensive rats. No differences in renal kallikrein messenger RNA (mRNA) levels were found between adrenalectomized rats and those treated for 5-14 days with 9 alpha-fludrocortisone, corticosterone or dexamethasone, or between hypertensive rats and their appropriate controls. Since mRNA levels appear essentially invariant under such circumstances, the change in renal kallikrein activity/immunoreactivity after chronic mineralocorticoid elevation, or in hypertensive rats, presumably reflects modulation at the post-transcriptional level.

alpha-N-acetylated beta-endorphin is differentially processed in the normal and hypothalamo-pituitary-disconnected (HPD) ewe.

We have used specific radioimmunoassay and high-performance liquid chromatography (HPLC) to determine content and molecular forms of pituitary alpha-N-acetylated endorphin (NacEP) in the intact and hypothalamo-pituitary-disconnected (HPD) sheep. No significant differences were seen in anterior pituitary (AP) immunoreactive (ir-) NacEP levels (control = 4.34 +/- 0.45 ng/mg; HPD = 4.38 +/- 0.83 ng/mg; n = 4) or in HPLC profiles following pituitary disconnection. In contrast, levels of ir-NacEP in the HPD intermediate lobe (IL) (263 +/- 16.5 ng/mg; n = 4) were double those in the control (110 +/- 14 ng/mg; n = 4), accompanied by a marked change in the molecular profile. In the control IL, the major species were Nac alpha EP (30%), Nac gamma EP (30%) and Nac beta EP1-27 (35%); in HPD IL, Nac beta EP1-27 (65%) is the dominant molecular form, with Nac alpha EP (11%) and Nac gamma EP (9%) relatively minor components.

Vasopressin-neurophysin II gene expression in the ovary: studies in Sprague-Dawley, Long-Evans and Brattleboro rats.

The neurohypophysial hormones oxytocin and arginine vasopressin (AVP) have been identified on immunological criteria in the ovary. Confirmation of extraneuronal synthesis requires the demonstration in the tissue of the specific messenger RNA (mRNA) for the preprohormone. Using a synthetic pentadecamer nucleotide probe, highly specific for the 5' region of rat neurophysin II (NP II), we have demonstrated the presence of AVP-NP II mRNA in the ovary of Sprague-Dawley, Long-Evans and Brattleboro rats, with an apparent molecular weight identical to that seen for hypothalamus. These findings, together with the presence of immunoreactive AVP in the ovaries but not hypothalami of Brattleboro rats, suggest that tissue-specific differences in AVP-NP II gene expression occur at the translational as well as transcriptional level.