Pituitary Venous Blood Research Articles

A comparison between the equine and bovine hypothalamus-pituitary-adrenocortical axis

In this review, we address the function of the hypothalamus-pituitary-adrenocortical (HPA) axis with special emphasis on the comparison between the bovine and equine species. The pars intermedia of the pituitary gland is particularly well developed in horses and cattle. However, its function is not well appreciated in cattle yet. The Wulzen's cone of the adenohypophysis is a special feature of ruminants. Total basal cortisol concentration is much higher in horses than that in cows with similar free cortisol fractions. Corticotropin-releasing factor (CRF) concentrations in equine pituitary venous blood are lower compared with other species, whereas plasma ACTH concentrations in cows are higher than those in horses. A CRF challenge test induced a more pronounced cortisol response in horses compared with cattle, whereas regarding ACTH challenge testing, the opposite seems true. Based on data from literature, the bovine species is characterized by relatively high basal blood CRF and ACTH and low cortisol and glucose concentrations. Obviously, further lowering of blood cortisol in cattle is easily prevented by the high sensitivity to ACTH, and as a consequence, subsequent increased gluconeogenesis prevents imminent hypoglycemia. Hypoglycemia is less likely in horses given their high muscle glycogen content and their relatively high cortisol concentration. When assessing HPA axis reactivity, response patterns to exogenous ACTH or CRH might be used as a reliable indicator of animal welfare status in cows and horses, respectively, although it is emphasized that considerable caution should be exercised in using measures of HPA activity solely to assess animal welfare.

Maximizing the accuracy of Inferior petrosal sinus sampling: validation of the use of Prolactin as a marker of pituitary venous effluent in the diagnosis of Cushing’s disease

This study aimed to increase the accuracy of the inferior petrosal sinus sampling (IPSS) procedure and reduce the false-negative rate through the addition of prolactin as a marker of pituitary venous outflow as well as validate this adjunct to the test process. Inferior petrosal sinus sampling (IPSS) for ACTH is the current gold standard test for the differentiation of pituitary Cushing's disease from the ectopic ACTH syndrome. Although early studies with IPSS reported a diagnostic sensitivity and specificity approaching 100%, additional experience has revealed a false-negative rate of 1-10%. This has been attributed to either technical problems with unsuccessful petrosal sinus catheterization or anomalous venous drainage of the pituitary. Previous studies have suggested that the measurement of other anterior pituitary hormones may be useful during IPSS as a guide to the effectiveness of cannulation and to improve the diagnostic accuracy of the procedure. We reviewed the data, in this retrospective cohort study, for all patients who had undergone IPSS for the investigation of ACTH-dependent hypercortisolism. The study included 83 patients who underwent IPSS at St. Thomas's hospital between 2005 and 2010. Plasma ACTH and prolactin levels were measured both centrally and peripherally. The normalized ACTH/Prolactin inferior petrosal sinus/peripheral ratio was then calculated to assess the accuracy of the sampling procedure. A total of 83 patients with confirmed ACTH-dependent cortisol excess underwent investigation with IPSS during the study period. Sixty-seven patients initially had a positive IPSS result (i.e. a basal central/peripheral ACTH ratio >2·0 and >3·0 post-CRH). However, when the concurrent prolactin data were used, six patients were additionally found to have positive results suggestive of pituitary Cushing's. The Prolactin normalized ACTH IPS/Peripheral ratios were all >0·8 in patients with proven Cushing's disease, whereas they were all <0·6 in proven ectopic ACTH syndrome. The diagnosis was subsequently confirmed histologically in 72 of the patients. Using Prolactin as a concurrent index of pituitary venous effluent helps us recognize whether pituitary venous blood has been accurately sampled. Normalizing the IPS/peripheral ratios with Prolactin helps to improve the accuracy of the result and reduces the false-negative rate. With regards to the usefulness/validity of this test in clinical practice, it is relevant, reproducible and is easily adaptable from the existing diagnostic sequence.

Inter‐Relationships Between the Secretory Dynamics of Thyrotrophin‐Releasing Hormone, Thyrotrophin and Prolactin in Periovulatory Mares: Effect of Hypothyroidism

We used our nonsurgical technique for collecting pituitary venous blood to relate the dynamics of thyrotrophin-releasing hormone (TRH) secretion to the secretion patterns of both prolactin and thyrotrophin in periovulatory mares, either euthyroid (n = 5) or made hypothyroid by treatment with propyl-thiouracil (n = 5). Pituitary venous blood was collected continuously and divided into 1-min aliquots for 4 h. To test the effect of dopamine on the relationship between secretion patterns, sulpiride, a selective D2 receptor antagonist, was given i.m. after 2 h of sampling. Thorough testing of the model and blood collection procedure revealed no sites of TRH loss. Hypothyroidism increased the mean secretion rates of TRH (P = 0.04) and thyrotrophin (P < 0.0001) but not prolactin. Sulpiride increased prolactin secretion rates in hypothyroid (P < 0.0001) and control (P = 0.007) mares, but did not alter TRH or thyrotrophin secretion rates. In both groups of mares, all three hormones were secreted episodically but not rhythmically. In both groups, the secretion pattern of TRH was almost always significantly related to that of thyrotrophin, as assessed by cross correlation and cross approximate entropy (ApEn) analysis. However, the degree of linear correlation was weak, with only 14% (hypothyroid) or 8% (controls) of the variation in thyrotrophin secretion rates attributable to TRH. Prolactin and TRH secretion patterns before sulpiride were coupled on cross ApEn analysis in both groups, and the minute-to-minute secretion rates of the two hormones were correlated in four hypothyroid and three euthyroid mares. Overall, the small, but significant, degree of association between TRH and prolactin was similar to that between TRH and thyrotrophin. In hypothyroid mares, sulpiride increased (P = 0.02) the synchrony between TRH and prolactin patterns. We conclude that in horses: (i) little TRH degradation occurs during passage through the pituitary or in blood after 1 h at 37 degrees C; (ii) TRH is not the major factor controlling minute-to-minute fluctuations in either thyrotrophin or prolactin; and (iii) reducing two strongly inhibitory inputs (i.e. dopamine and thyroid hormones) may magnify the stimulatory effect of TRH on prolactin secretion.

The effect of endotoxin administration on the secretory dynamics of oxytocin in follicular phase mares: relationship to stress axis hormones.

The primary aim of this study was to define the secretory dynamics of oxytocin and vasopressin in pituitary venous effluent from ambulatory horses during acute endotoxaemia, a stimulus that may release both hormones. Our secondary aim was to investigate the role of oxytocin in regulating adrenocorticotropic hormone (ACTH) secretion by comparing oxytocin, vasopressin, corticotropin-releasing hormone (CRH) and ACTH secretory profiles during endotoxaemia and by monitoring the ACTH response to oxytocin administration. Pituitary venous blood was collected nonsurgically continuously and divided into 1-min segments from eight follicular phase mares. Four mares were sampled for 30 min before and 3.5 h after receiving an i.v. infusion of bacterial endotoxin (TOX). Four control mares were sampled for 2.5 h without infusion of TOX. Another three follicular phase mares were given 5 U of oxytocin to replicate the peak response to TOX and pituitary blood collected every 1 min for 10 min before and 15 min after injection. Endotoxin raised the secretion rates of all hormones measured. All hormones were released episodically throughout the experiment, with TOX increasing the amplitude of peaks in each hormone. Peaks in oxytocin and vasopressin were coincident in each treated mare. Similarly, ACTH peaks were coincident with peaks of oxytocin and vasopressin in each treated mare, and with peaks of CRH in three mares. However, oxytocin administration did not affect ACTH secretion. We conclude that during endotoxaemia in horses: (i) oxytocin and vasopressin are secreted synchronously; (ii) oxytocin is unlikely to be acting as an ACTH secretagogue since inducing peak oxytocin concentrations observed during TOX does not raise ACTH; and therefore (iii) the close relationship between oxytocin and ACTH secretion is circumstantial and due to the fact that oxytocin secretion is concurrent with that of vasopressin, a proven ACTH secretagogue in horses.

The effect of the alpha-2-adrenergic agonist, clonidine, on secretion patterns and rates of adrenocorticotropic hormone and its secretagogues in the horse.

Alpha-2-adrenoceptor activation may lower adrenocorticotropic hormone (ACTH) by reducing secretagogue input and/or increasing the release of an inhibitory factor (CIF). To investigate this, we gave clonidine, an alpha-2-agonist, to seven horses, and collected pituitary venous blood every minute for 20 min before treatment and 40 min after treatment. Six horses were given saline vehicle. Mean secretion rates of corticotrophin-releasing hormone (CRH), arginine vasopressin (AVP) and ACTH were calculated before and during four 5-min then two 10-min periods after clonidine or saline. Reduction in ACTH secretion without corresponding changes in CRH and/or AVP would imply the presence of CIF. Secretion rates of ACTH (P = 0.008) and AVP (P = 0.0005) fell after clonidine and remained lower than baseline values for 20 min and 10 min, respectively. The CRH secretion rate decreased slightly but not significantly after clonidine. In controls, hormone secretion rates did not alter during the experiment. Multiple linear regression showed that CRH and AVP secretion accounted for 69% (treated) or 45% (controls) of the variation in ACTH secretion (P < 0.0001 for each). CRH alone contributed 80% (treated) or 76% (controls) of the fit to this model, which is consistent with the concept that CRH 'sets the gain' of the response of corticotrophs to fluctuations in AVP. Accordingly, minute-to-minute changes in pituitary concentrations of AVP and ACTH were synchronous when all data were considered (% concordant changes: controls, 68%, P < 0.0001; treated, 76%, P < 0.0001) and the percentage of concordant movement was unaffected by clonidine (before 72%; after 73%; P = 0.80). In treated horses but not controls, the ratio between the secretion rates of ACTH and AVP fell (P = 0.009), while the ACTH : CRH ratio tended to fall after clonidine, implying reduced responsiveness to stimulation. Moreover, one horse showed a drop in ACTH and a rise in CRH and AVP secretion after clonidine. We conclude that in horses alpha-2-adrenoceptor activation lowers ACTH secretion primarily by reducing the secretion of AVP and possibly CRH. While there was some evidence that a CIF may participate in the clonidine-induced suppression of ACTH, the subtlety of the discordance between ACTH and its secretagogues in most horses and the rarity of complete dissociation indicate that it does not play a major role.

Effect of insulin-induced hypoglycaemia on secretion patterns and rates of corticotrophin-releasing hormone, arginine vasopressin and adrenocorticotrophin in horses.

To study the effect of hypoglycaemia on secretion rates of corticotrophin-releasing hormone (CRH), arginine vasopressin (AVP) and ACTH in a non-ruminant species, a non-surgical method was used to collect pituitary venous (PitVen) blood every 0.5 or 1 min from seven horses before and after insulin administration (0.4 U/kg i.v.). To assess the effect of PitVen cannulation on results, peripheral hormones were also measured before and after insulin in five horses without PitVen cannulae. Insulin administration lowered plasma glucose in all horses (P < 0.0001; paired t-test). Cortisol concentrations, which were similar in horses with and without PitVen cannulae before insulin, rose significantly after insulin administration in both groups. Most horses showed discomfort as glucose fell. When data from horses with and without PitVen cannulae were pooled, the peak fractional change in cortisol (Spearman's rank correlation coefficient (rs) = -0.94, P < 0.001) and the severity of hypoglycaemic symptoms (rs = -0.61, P < 0.02) were inversely ranked with the glucose nadir. In horses with PitVen cannulae, insulin administration increased secretion rates of ACTH (P < 0.0001), AVP (P < 0.0001) and CRH (P < 0.02). Increments in ACTH (rs = -0.96, P < 0.005) and CRH (rs = -0.81, P < 0.05), but not in AVP, measured during the second half-hour after insulin (i.e. the peak response), were inversely ranked with the glucose nadir. Moreover, ACTH increments were positively ranked with those in CRH (rs = 0.81, P < 0.05), but not in AVP. Nevertheless, in individual horses, minute-to-minute AVP and ACTH concentrations in PitVen blood were always correlated, whereas minute-to-minute CRH and ACTH concentrations were correlated only when glucose dropped below 3.4 mmol/l. In less hypoglycaemic horses, ACTH secretion rose despite little or no change in CRH. We suggest that in horses AVP is the primary acute signal for ACTH release both before and during hypoglycaemia; however, the increasing magnitude of ACTH increments induced by greater degrees of hypoglycaemia is determined largely by selective CRH release, which then augments corticotroph responses to AVP.

Patterns of secretion of GnRH, LH and FSH during the postovulatory period in mares: mechanisms prolonging the LH surge.

To study the mechanisms responsible for the unusually slow decline of the ovulatory LH surge in mares, secretion patterns of GnRH, LH and FSH were monitored in pituitary venous blood collected every 2 or 5 min for 10.5-18.0 h from five mares on the third (n = 4) or fifth day after ovulation (first sampling period). To determine the effectiveness of progesterone negative feedback, mares were then given a luteolytic dose of a prostaglandin analogue (PGF2 alpha) and pituitary venous sampling (every 2 or 5 min for 16 h) recommenced 20-22 h later (second sampling period). During the declining arm of the LH surge, large peaks (detected by the Cluster algorithm) of concurrent LH and FSH secretion occurred infrequently, with four peaks being detected in a combined sampling period of 75 h. Outside the peaks, LH or FSH secretion continued (as assessed by a pituitary to jugular-venous concentration ratio > or = 1.25) during 46% +/- 13 or 40% +/- 10, respectively, of the sampling period. GnRH immunoactivity was detected during each spontaneous gonadotrophin peak, but at other times was generally at assay sensitivity. After PGF2 alpha, plasma progesterone fell (ng ml-1, mean +/- SEM; first sampling period: 8.6 +/- 0.8; second; 2.0 +/- 0.3; P = 0.001) and the frequency of LH (P < 0.05) and FSH (P < 0.02) peaks rose, with 28 peaks detected for each hormone in a total of 80 h sampling. Peaks in LH were smaller during the second period, with decreases observed in maximum (P = 0.027) and mean (P = 0.025) secretion rates. Maximum GnRH secretion rate during peaks also declined (P = 0.010); however, the decrement (-30 +/- 6%) was less than that in maximum LH secretion rate (-82 +/- 5%; P = 0.040), suggesting that other factors contribute to the reduced LH peak amplitude. In summary, gonadotrophin peak frequency during the downswing of the surge in mares is slow, as in the midluteal phase, and the slow rate of decline in peripheral gonadotrophin concentrations is due, at least in part, to continued secretion between pulses. Moreover, progesterone negative feedback is highly effective in early dioestrus, in that lessening it without complete removal markedly accelerates gonadotrophin pulse frequency.

GnRH secretion in the mare

We review our research on the regulation of the mare's ovulatory cycle. We describe the dynamics of GnRH, LH and FSH secretion during the cycle as determined by collecting pituitary venous blood and discuss the central mechanisms involved in generating an ovulatory LH surge. We also consider protocols giving GnRH or its analogues to induce ovulation out-of-season and relate results to the normal control of gonadotropin secretion.

Dynamics of the Regulation of the Hypothalamo–Pituitary–Adrenal (HPA) Axis Determined Using a Nonsurgical Method for Collecting Pituitary Venous Blood from Horses

Since 1985, we have applied our nonsurgical technique for collecting pituitary venous (PitVen) blood from ambulatory horses to investigate the regulation of adrenocorticotropic hormone (ACTH) secretion. This method offers particular advantages for studying the hypothalamo–pituitary–adrenal axis since its benign nature enables hypothalamic and pituitary interactions to be monitored without disturbing the animal, and the horse’s large blood volume allows 3- to 4-ml samples to be collected as frequently as every 20 s for prolonged periods so that the secretion patterns of ACTH and its secretagogues can be precisely defined. When PitVen blood was sampled every 20 or 30 s during the circadian maximum, arginine vasopressin (AVP) and ACTH secretion patterns were complex and irregular, with mean interpeak intervals of approximately 5 min. Despite their erratic patterns, AVP and ACTH secretions were closely coupled on cross-correlation analysis. By contrast, PitVen corticotropin-releasing hormone (CRH) concentrations were low, relatively stable, and not consistently related to ACTH secretion. However, when cortisol negative feedback was reduced acutely by metyrapone infusion, CRH and AVP secretion were stimulated. Mathematical modeling suggested that CRH had become the more effective secretagogue and that much of the ACTH response was mediated by increased pituitary responsiveness to CRH. Elevated blood osmolality triggered synchronous AVP and ACTH secretion, without altering PitVen CRH. In this case, the source of PitVen AVP was presumably the magnocellular/neurohypophysial pathway, which is thought to respond primarily to changes in blood osmolality and pressure. Our results suggest that this pathway also participates in ACTH regulation. We have studied the effect of several perturbations and found, as have others, that the secretagogues released vary with the stimulus given. For example, vigorous exercise promptly raised PitVen AVP and ACTH, but not PitVen CRH. Hypoglycemia provoked both CRH and AVP secretions, with the CRH increment being inversely proportional to the glucose nadir. Administration of the opioid antagonist, naloxone, increased PitVen ACTH; however, changes in AVP and CRH were variable and overall could not account for the ACTH response. This suggests that endogenous opioids inhibit a third ACTH secretagogue, stimulate an inhibitory factor, or also act at the pituitary. Chronic social stress, induced by confining newcomers with aggressive, resident mares, caused most introduced horses to become submissive. In such horses, plasma cortisol declined to levels similar to those during metyrapone infusion. Despite hypocortisolemia, PitVen ACTH was low, whereas PitVen CRH tended to be elevated. Moreover, chronically stressed horses did not respond to exogenous CRH. We conclude that at rest and during some perturbations AVP is the immediate stimulus for ACTH release. Even ACTH micropulses, previously thought to occur spontaneously, appear to be regulated by AVP in horses. On the other hand, CRH secretion and pituitary responsiveness to CRH rise when cortisol falls, suggesting that a major role for CRH is to fix the cortisol setpoint. However, during chronic stress, these relationships become disturbed, with results to date pointing toward the existence of an ACTH-release inhibiting factor.

The effect of naloxone administration on the secretion of corticotropin-releasing hormone, arginine vasopressin, and adrenocorticotropin in unperturbed horses.

We used our nonsurgical method for collecting equine pituitary venous blood to study the role of endogenous opioids in the basal regulation of the hypothalamo-pituitary-adrenal axis. We gave mares the opioid antagonist, naloxone (NAL), at either a high (0.5 mg/kg i.v. bolus, followed by infusion of 0.25 mg/kg.h; n = 4) or low (0.2 mg/kg i.v. bolus; n = 6) dose rate. Pituitary venous blood was collected continuously, divided into 0.5- or 1-min segments for 15-30 min before and 1 h after the NAL bolus, and assayed for CRH, arginine vasopressin (AVP), and ACTH. The mares tolerated NAL administration well, with little difference between dose rates in the mild transient side-effects. Both NAL doses increased jugular cortisol concentrations (high, P = 0.0022; low, P = 0.0001) and the ACTH secretion rate (high, P = 0.0056; low, P = 0.0103). High dose NAL raised the secretion rates of AVP (P = 0.0252) and CRH (P = 0.0106); however, the magnitude of ACTH responses exceeded those in AVP and CRH, as shown by increased ratios between ACTH and AVP (P = 0.0246) or CRH (P = 0.0122) secretion rates. After low dose NAL, neither CRH nor AVP secretion was altered. Indeed, CRH declined as ACTH rose in 4 mares and was unchanged in a fifth mare. When data from the 10 mares were pooled, mean secretion rates of ACTH and CRH were correlated after (P < 0.05), but not before, NAL treatment. Overall, mean ACTH and AVP secretion rates were not correlated during any 30-min period, but in individual mares, minute to minute AVP and ACTH secretion patterns were always correlated. We conclude that endogenous opioids inhibit the equine hypothalamo-pituitary-adrenal axis under basal conditions; however, their sites of action do not appear to lie solely on CRH and/or AVP neurons. It seems likely that endogenous opioids also inhibit the release of a third ACTH secretagogue or promote the secretion of an ACTH release inhibitory factor.

Oxytocin Secretion Patterns in Normal Stallions as Measured in Pituitary Venous Blood: Correlation with Gonadotropin Secretion and Effect of Sexual Arousal1

Oxytocin Secretion Patterns in Normal Stallions as Measured in Pituitary Venous Blood: Correlation with Gonadotropin Secretion and Effect of Sexual Arousal1

The dynamics of gonadotrophin-releasing hormone, LH and FSH secretion during the spontaneous ovulatory surge of the mare as revealed by intensive sampling of pituitary venous blood

Conflicting views exist on the mode of gonadotrophin-releasing hormone (GnRH) secretion during the ovulatory LH surge and the relative importance of changes in pituitary responsiveness to GnRH in generating the LH surge. This disagreement may stem from species differences and/or methodological problems. To provide data on the exact relationship between GnRH and gonadotrophin secretion during the spontaneous LH surge, we collected pituitary venous (PV) blood every 30 s for 3-4 h from eight mares and then assayed GnRH (in six of the mares), FSH and LH. Jugular blood was also collected from twelve mares without PV cannulae either thrice daily during the surge (n = 8) or hourly for 24 h when close to ovulation (n = 4) and assayed for LH. Hormone peaks in PV blood were detected by the Cluster program and PV hormone patterns were scanned for underlying periodicity using spectral analysis. Jugular LH concentrations rose slowly and steadily without abrupt increase during the prolonged ovulatory surge, suggesting that hormone secretory patterns seen during the periods of rapid sampling were typical of the surge. Jugular LH concentrations were similar in mares with and without PV cannulae. Intensive sampling of PV blood showed that GnRH, FSH and LH were secreted in frequent (two to five per h) brief (5-7 min) peaks. Secretion was not detectable in 24%, 28% and 57% of the total sampling time for GnRH, LH and FSH respectively. GnRH and LH peaks appeared to be irregular in time and amplitude in most mares. However, spectral analysis of the data revealed an underlying periodicity in the secretion of all three hormones, with the dominant period ranging from 20 to 65 min in individual mares. The spectra of GnRH, FSH and LH were highly coherent at this dominant frequency, and 90% of GnRH peaks were concurrent with LH peaks, which is consistent with the dogma that GnRH is the primary secretagogue for both FSH and LH. Although PV FSH and LH concentrations were closely correlated, PV GnRH and gonadotrophin concentrations were only weakly correlated, implying that there was no consistent relationship between the magnitudes of changes in GnRH and gonadotrophin secretion. When compared with our published mid-luteal phase values, the daily GnRH secretion rate during the LH surge was trebled, while the LH responsiveness to endogenous GnRH, as assessed by the ratio between newly secreted LH and PV GnRH concentrations, was four times greater.(ABSTRACT TRUNCATED AT 400 WORDS)

Short-term secretion patterns of corticotropin-releasing hormone, arginine vasopressin and ACTH as shown by intensive sampling of pituitary venous blood from horses.

To characterize the short-term ACTH secretion pattern and to investigate factors regulating it, pituitary venous (PV) blood was collected using our nonsurgical method from 8 unperturbed horses every 20 or 30 s for approximately 1 h. In all but 1 horse, sampling occurred during the broad circadian maximum in plasma cortisol concentrations. Concentrations of corticotropin-releasing hormone (CRH; n = 7 horses), arginine vasopressin (AVP), ACTH and cortisol were measured by radioimmunoassay. In all horses, CRH, AVP and ACTH secretion patterns appeared irregular in time and amplitude. The mean (+/- SEM) numbers of peaks per hour detected by the cluster program were 2.8 +/- 1.2, 10.1 +/- 1.9 and 10.2 +/- 1.4 for CRH, AVP and ACTH, respectively. However, when 2- and 5-min sampling frequencies were simulated by meaning consecutive values, significantly fewer peaks were detected in each hormone. There was no correlation between the prevailing cortisol concentration and peak frequencies of CRH, AVP or ACTH. Secretion patterns of ACTH and AVP were closely related in all horses as assessed by cross correlation analysis and coincidence of peaks, although the ratio between PV ACTH and AVP concentrations fluctuated markedly within each horse. In contrast, the relationship between CRH and ACTH secretion was variable. Bivariate spectral analysis showed only a modest degree of underlying periodicity in CRH, AVP and ACTH secretion during the very short term studied. Nevertheless, distinct peaks exceeding the 95% confidence limits of white noise were observed at periods between 2 and 30 min in 5 of 7 CRH, 6 of 8 AVP and 5 of 8 ACTH spectra. Furthermore, the slope of the regression line through each spectrum did not become indistinguishable from zero, i.e. the flat white noise continuum, until mean (+/- SEM) periods of 2.6 +/- 0.8, 1.6 +/- 0.2, and 2.0 +/- 0.2 min, for CRH, AVP and ACTH spectra, respectively. At the ACTH spectral maximum, the coherence coefficient, which is analogous to the squared correlation coefficient, exceeded 0.5 in comparisons of all ACTH and AVP spectra and of 5 of 7 ACTH and CRH spectra.(ABSTRACT TRUNCATED AT 400 WORDS)

The acute effect of lowering plasma cortisol on the secretion of corticotropin-releasing hormone, arginine vasopressin, and adrenocorticotropin as revealed by intensive sampling of pituitary venous blood in the normal horse.

The effect of an acute fall in plasma cortisol on the secretion of CRH, arginine vasopressin (AVP), and ACTH was studied using our nonsurgical technique for collecting pituitary venous (PV) blood from horses. PV blood from six mares was collected continuously and divided into 30-sec segments for 0.5 h before and during a 3-h infusion of metyrapone, an 11-beta-hydroxylase inhibitor. During treatment, plasma cortisol fell (P < 0.01) to a mean nadir of 15% of pretreatment levels, and 11-deoxy-cortisol rose (P < 0.02). Three mares became mildly agitated during treatment. Mean PV concentrations of CRH (P < 0.025), AVP (P < 0.05), and ACTH (P < 0.005) were higher during the second hour of treatment than before. For AVP (P < 0.05) and ACTH (P < 0.01), the amount secreted in peaks detected by CLUSTER analysis increased during treatment, whereas peak frequency did not. Responses, particularly in CRH and AVP, tended to be amplified during agitation. Increases in CRH, AVP, and ACTH secretion commenced when cortisol had fallen to 50-59% of the initial value (P < 0.005 for each). By contrast, the cortisol concentration at this point varied 3-fold among mares. The ratio between PV concentrations of ACTH and CRH, which was used as an index of pituitary responsiveness to endogenous CRH, also rose (P < 0.005) as cortisol fell. The increase in this ratio preceded any significant change in CRH secretion and was maintained to the end of the experiment. We suggest that the initial response to falling cortisol in the horse is at the pituitary, via increased responsiveness to CRH. If cortisol continues to fall, AVP and then CRH secretion are stimulated. However, the magnitude of the hypothalamic response to hypocortisolemia may be augmented by concurrent stress. Last, the hypothalamo-pituitary-adrenal axis of the horse appears to monitor changes in plasma cortisol and not concentrations, at least in the short term.

The acute effect of lowering plasma cortisol on the secretion of corticotropin-releasing hormone, arginine vasopressin, and adrenocorticotropin as revealed by intensive sampling of pituitary venous blood in the normal horse

The acute effect of lowering plasma cortisol on the secretion of corticotropin-releasing hormone, arginine vasopressin, and adrenocorticotropin as revealed by intensive sampling of pituitary venous blood in the normal horse

Secretory patterns and rates of gonadotropin-releasing hormone, follicle-stimulating hormone, and luteinizing hormone revealed by intensive sampling of pituitary venous blood in the luteal phase mare.

We used our unique nonsurgical technique for collecting pituitary venous (pit) blood to study GnRH, FSH, and LH secretion patterns in midluteal phase mares. This method does not perturb endocrine function and allows continuous monitoring of GnRH and gonadotropin (Gn) secretion, determination of the amount of GnRH perfusing gonadotropes, and direct measurements of the amounts of Gn secreted. In a total of 80 h of 5-min sampling in four mares, eight Gn peaks occurred; however, more frequent sampling was needed to define secretory events precisely. Therefore, pit blood was collected continuously and split into 30-sec segments in six mares. To ensure a peak during sampling, the opioid antagonist naloxone was given after 4-6 h of sampling to try to replicate a physiological signal for GnRH release. Naloxone induced Gn peaks in jugular blood that were indistinguishable in amplitude from spontaneous peaks. Intensive sampling of pit blood showed that jugular peaks reflected major episodes of GnRH and Gn secretion lasting 30-55 min, which were similar in profile whether naloxone induced or spontaneous and consisted of a train of three to six peaks of diminishing amplitude. Peaks of GnRH and, less often, Gn also occurred outside major episodes. Despite markedly variable size, GnRH peak maxima were correlated with the amount of LH and FSH secreted in concurrent peaks. Likewise, cross-correlation analyses (n = 960 samples/mare) showed close correspondence between patterns of GnRH and secreted FSH and LH. The delay (+/- SEM) between GnRH and Gn maxima was 0.62 +/- 0.18 min for LH and 0.18 +/- 0.22 min for FSH. The majority of GnRH and Gn peaks were concurrent; however, 34.7% of GnRH peaks occurred without Gn peaks. These peaks had a lower amplitude than those with Gn peaks (P < 0.001). For Gn, secretion (i.e. ratio between pit and jugular concentrations, > 1.5) continued at a low level for 40 +/- 9% (LH) or 64 +/- 14% (FSH) of the time between Cluster-defined peaks during the basal period. We conclude that in the luteal phase 1) the predominant mode of GnRH and Gn secretion is as concurrent, large amplitude, prolonged episodes that appeared to be the summation of a train of peaks; and 2) a GnRH dose-Gn response relationship operates endogenously. This along with the synchronicity of secretion patterns of the three hormones suggest that GnRH is the major secretagogue for both LH and FSH.

Secretory patterns and rates of gonadotropin-releasing hormone, follicle-stimulating hormone, and luteinizing hormone revealed by intensive sampling of pituitary venous blood in the luteal phase mare

Secretory patterns and rates of gonadotropin-releasing hormone, follicle-stimulating hormone, and luteinizing hormone revealed by intensive sampling of pituitary venous blood in the luteal phase mare

The Effect of Acute Exercise on the Secretion of Corticotropin-Releasing Factor, Arginine Vasopressin, and Adrenocorticotropin as Measured in Pituitary Venous Blood from the Horse

We have used the technique which we have developed for collecting pituitary venous blood from conscious, undisturbed horses to study the effect of acute vigorous exercise on the secretion of CRF, arginine vasopressin (AVP) and ACTH. Pituitary venous (pit) blood was collected every 1-5 min from nine trained racehorses at rest in the stable. The horses then trotted quietly for 10 min, after which they galloped as fast as possible for 4-6 min, before returning to the stable where sampling continued. In Exp 1 (n = 5) no blood samples were taken during exercise, whereas in Exp 2 (n = 4), pit blood was collected every 30 sec during exercise. Immediately after exercise, significant elevations in heart rate (P less than 0.001), body temperature (P less than 0.01) and hematocrit (P less than 0.001) were observed as compared with preexercise values. Jugular cortisol levels were higher after exercise (301.9 +/- 35.2 nmol/liter; mean +/- SEM) than before (187.3 +/- 34.8; P less than 0.01; n = 9). Likewise, jugular AVP levels increased with exercise (before, 0.65 +/- 0.11 pmol/liter; after 3.2 +/- 0.6; P less than 0.01; n = 6), whereas jugular CRF was not altered by exercise (before, 0.38 +/- 0.08 pmol/liter; after, 0.93 +/- 0.31; n = 6; NS). In Exp 1, no significant changes in pit ACTH, AVP, or CRF were observed after exercise. However in Exp 2 when pit blood was sampled during exercise all horses showed an immediate and dramatic rise in ACTH (P less than 0.01) and AVP (P less than 0.005) secretion which peaked during galloping with mean fractional changes above resting levels of 23.6 +/- 9.9 for ACTH and 51.7 +/- 24.0 for AVP. After exercise pit AVP levels were not different from resting, whereas ACTH remained elevated (11.4 +/- 6.9-fold above resting levels). By contrast, pit CRF levels were not altered by exercise. In both experiments together, pit AVP and ACTH concentrations were correlated in eight of the nine horses, whereas pit CRF and ACTH concentrations were positively correlated in only one of seven horses. We conclude that acute exercise causes a transient increase in ACTH secretion which occurs synchronously with an increase in AVP secretion. CRF does not appear to play a major role in mediating the initial ACTH response to exercise.

Effect of an Osmotic Stimulus on the Secretion of Arginine Vasopressin and Adrenocorticotropin in the Horse*

Arginine vasopressin (AVP) is released in response to changes in blood osmolality and is also a putative secretagogue for ACTH. However, it is unclear whether osmotically generated increases in AVP in the physiological range influence ACTH secretion. We have studied this question using our unique noninvasive technique for collecting pituitary venous blood in six normal conscious horses that received an iv infusion of hypertonic saline (HS; 5%, 0.07 ml/kg.min) for 45-60 min. Pituitary and jugular venous samples were collected every 5 min for 40 min before, during, and for 20 min after HS. During HS, mean blood osmolality rose (P less than 0.01), with a mean peak increase of 14.8 mosmol/kg (range, +6-+37 mosmol/kg). Jugular AVP rose (P less than 0.01) from 0.56 +/- 0.18 pmol/liter (mean +/- SEM) before HS to 2.16 +/- 0.86 pmol/liter during HS. Mean jugular AVP and osmolality were correlated (r = 0.82; P less than 0.05) during HS. Mean jugular ACTH concentrations increased (P less than 0.01) from 49 +/- 9 ng/liter before HS to 148 +/- 54 ng/liter during HS, while mean cortisol levels during and after HS exceeded basal levels (P less than 0.05). Pituitary AVP and ACTH concentrations exceeded jugular concentrations by up to 100-fold, and mean (P less than 0.01 for both) and peak (P less than 0.001 for both) levels increased during HS. AVP and ACTH secretion during HS were pulsatile. The mean and peak changes in pituitary AVP were significantly correlated with those in ACTH. For the six horses together, pituitary ACTH and AVP concentration changes occurred synchronously during the experiment (P less than 0.001), and the paired AVP and ACTH concentrations were highly correlated (r = 0.73; n = 129 pairs; P less than 0.001). We conclude that 1) physiological changes in AVP secretion are closely associated with comparable changes in ACTH secretion, and 2) osmotic signals that presumably activate the magnocellular neurons of the supraoptic and paraventricular nuclei may be physiologically relevant regulators of corticotrope function.

The effects of cortisol, vasopressin (AVP), and corticotropin-releasing factor administration on pulsatile adrenocorticotropin, alpha-melanocyte-stimulating hormone, and AVP secretion in the pituitary venous effluent of the horse.

Plasma ACTH, arginine vasopressin (AVP), and alpha MSH were measured in pituitary venous effluent at 5-min intervals from five unanesthetized horses during cortisol infusion and after an iv bolus of AVP or ovine (o) CRF. In control experiments (no hormone) there was a significant overall correlation between the timing of concentration changes in ACTH and alpha MSH. Cortisol infusion increased jugular cortisol levels by 70% and was associated with a reduction in mean ACTH, AVP, and alpha MSH secretion rates and ACTH peak secretion rate, but did not alter the observed pulse frequencies of these hormones. Administration of AVP raised plasma concentrations to a level comparable to the spontaneous peaks in pituitary venous blood and resulted in an increase in the secretion of ACTH and alpha MSH in all horses. Furthermore, spontaneous AVP peaks occurred in pituitary venous blood between 90 and 180 min after AVP injection, indicating that the exogenous hormone did not suppress AVP secretion. oCRF administration led to a prolonged elevation in plasma CRF and an increase in secretion of ACTH and alpha MSH, but not AVP, in all horses. The pulsatile secretion of ACTH and alpha MSH was maintained despite plasma CRF levels in excess of 400 pmol/liter, and the timing of concentration changes in AVP and ACTH continued to be highly correlated. It is concluded that pulsatile ACTH secretion continues during cortisol, oCRF, or AVP administration. Like that of ACTH, alpha MSH secretion is stimulated by oCRF and AVP administration and suppressed by cortisol. Although the timing of concentration changes in ACTH and alpha MSH is highly correlated, the correlation of the actual concentrations of these two hormones varies considerably in different animals.