Search Results

You are looking at 11 - 18 of 18 items for

  • Author: R A Donald x
  • Refine by access: All content x
Clear All Modify Search
M J Evans
Search for other papers by M J Evans in
Google Scholar
PubMed
Close
,
R S Mulligan
Search for other papers by R S Mulligan in
Google Scholar
PubMed
Close
,
J H Livesey
Search for other papers by J H Livesey in
Google Scholar
PubMed
Close
, and
R A Donald
Search for other papers by R A Donald in
Google Scholar
PubMed
Close

Abstract

Perifused equine anterior pituitary cells were used to investigate the relationships between the secretion of ACTH and substances known to either stimulate (corticotrophin-releasing hormone (CRH), and arginine vasopressin (AVP)) or inhibit (cortisol) ACTH secretion. The experiments were designed to mimic the hormone milieu present in vivo in the horse, with cortisol (0 or 100 nmol/l) and CRH (0 or 0·02 nmol/l) perifused continuously, and pulses of AVP (10 nmol/l) applied for 5 min at 30-min intervals.

In columns perifused with 0·02 nmol CRH/1 there was no significant overall effect of 100 nmol cortisol/l on the ACTH responses to pulses of AVP, although there was a significant interaction between AVP pulse number and cortisol showing that ACTH total area (pmol ACTH proportional to area under response curve) in response to AVP pulses 1 and 2 was significantly (P<0·05) decreased in columns perifused with 100 nmol cortisol/l. However ACTH incremental area (pmol ACTH proportional to the area above the CRH-induced baseline) was not affected by cortisol at any AVP pulse.

This contrasts with the effect of cortisol in columns perifused with 0 nmol CRH/l, where 100 nmol cortisol/l significantly decreased ACTH total area (P=0·0075) and incremental area (P=0·049) at all AVP pulses compared with the responses in columns receiving 0 nmol cortisol/l.

There was a fall off in ACTH responsiveness with time during the experiment which, in the presence of 0·02 nmol CRH/1, was significantly (P<0·001) greater with 0 nmol cortisol/l than with 100 nmol cortisol/l and if 6 (rather than 3) pulses of AVP were given, whereas with 0 nmol CRH/l there was no difference in the fall off with time between columns receiving 0 and 100 nmol cortisol/l.

These results show that the control of ACTH secretion is influenced not only by independent action of secretagogues such as CRH and AVP, or inhibitors such as cortisol, but by a complex interaction of these factors with one another. CRH may have a role in 'protecting' the ACTH response to pulses of AVP in the presence of cortisol. It follows that, in vivo, 'background' CRH could allow an increase in ACTH in response to AVP released by a new stress, despite the presence of elevated cortisol.

Journal of Endocrinology (1996) 148, 475–483

Restricted access
M J Ellis
Search for other papers by M J Ellis in
Google Scholar
PubMed
Close
,
R S Mulligan
Search for other papers by R S Mulligan in
Google Scholar
PubMed
Close
,
M J Evans
Search for other papers by M J Evans in
Google Scholar
PubMed
Close
, and
R A Donald
Search for other papers by R A Donald in
Google Scholar
PubMed
Close

Abstract

Antagonists are useful for probing hormone action and receptor characteristics. In this study we have investigated the inhibitory effects of analogues of arginine vasopressin (AVP) and corticotrophin-releasing hormone (CRH) on stimulated release of immunoreactive ACTH from perifused equine anterior pituitary cells in vitro. Our aims were to gain some insight into the characteristics of the CRH and AVP receptors of the horse pituitary and to establish whether the response induced by AVP and CRH together could be blocked by combining antagonists. Experimental design included 5-min pulses of AVP (12·5 nmol/l), CRH (0·3 nmol/l) or CRH plus AVP given every 40 min alternately with pulses of secretagogue(s) plus appropriate antagonist(s). The effect of combined antagonists on the response to lower secretagogue concentrations (CRH, 0·03 nmol/l plus AVP, 2·5 nmol/l) was also tested. Response in the presence of an antagonist was compared with the mean response to secretagogue in the immediately preceding and following pulse and was expressed as per cent expected ACTH.

The ACTH response to AVP was inhibited over the dose range 0·4–50 μmol/l by Phaa-d-Tyr(Et)2Lys6Arg3VP (P<0·002; ANOVA) and by d(CH2)5[Tyr(Me)2]AVP (P<0·001). Suppression of the expected ACTH response to AVP by these two antagonists was most effectively achieved by antagonist concentrations of 10 μmol/l (to 28±2·1%) and 25 μmol/l (to 22±5·1%) respectively. Inhibition was not improved by preinfusion compared with a bolus pulse. Aaa-d-Tyr(Et)2Val4Abu6Arg8·9VP and the non-peptide antagonist OPC-21268 had no inhibitory effect. Two α-helical (α-h) analogues of CRH, (α-hCRH(12–41) and α-hCRH(9–41) tested over the dose range 0·5–5 μmol/l, suppressed CRH-induced ACTH secretion (P<0·001) but CRH(23–41) had no significant effect. The α-hCRH(12–41) achieved greater suppression of ACTH release than the (9–41) derivative (8·7±4·2% compared with 19·3±3·5% of the expected ACTH response). Combination of d(CH2)5[Tyr(Me)2]AVP (25 μmol/l) plus α-hCRH (12–41) (5·0 μmol/l) achieved suppression to −0·5±1·3% and 0·8±1·5% of the expected response to CRH+AVP at 0·3+12·5 nmol/l and 0·03+2·5 nmol/l respectively. These effects were greater than seen by the individual antagonists alone.

The antagonist effects suggest that the CRH and AVP receptors of the equine pituitary have similar properties to those from other species and are consistent with the pituitary AVP receptor being unlike the V2 receptor and resembling but not being identical to the V1 type. We also conclude that α-hCRH(12–41) and d(CH2)5[Tyr(Me)2]AVP can together block the ACTH response to CRH plus AVP and suggest that these antagonists should provide a means of investigating additional secretagogues involved in ACTH release in the horse.

Journal of Endocrinology (1994) 143, 85–93

Restricted access
M. J. Evans
Search for other papers by M. J. Evans in
Google Scholar
PubMed
Close
,
A. G. Marshall
Search for other papers by A. G. Marshall in
Google Scholar
PubMed
Close
,
N. E. Kitson
Search for other papers by N. E. Kitson in
Google Scholar
PubMed
Close
,
K. Summers
Search for other papers by K. Summers in
Google Scholar
PubMed
Close
, and
R. A. Donald
Search for other papers by R. A. Donald in
Google Scholar
PubMed
Close

ABSTRACT

The multifactorial control of ACTH is well established. We wished to establish and characterize an in-vitro perifusion system, using equine anterior pituitary cells and physiological concentrations of secretagogues, to investigate factors which affect the dynamics of ACTH secretion. Anterior pituitary tissue was divided for dispersion into cells with collagenase, trypsin or dispase, or by mechanical dispersion. After dispersal followed by 18-h incubation, cells were perifused and the ACTH response to 10-min pulses of arginine vasopressin (AVP; 100 nmol/l), corticotrophin-releasing hormone (CRH; 0·01 nmol/l), and AVP (100 nmol/l) plus CRH (0·01 nmol/l) determined. ACTH responses to these secretagogues were lower (P <0·05) in cells prepared using the enzymes dispase and trypsin than with the enzyme collagenase. Cells prepared by mechanical methods were not responsive. Collagenase-prepared cells were used in subsequent experiments.

In dose-response studies (10-min pulse length), a steep CRH–ACTH dose-response curve was obtained with the minimum effective concentration of CRH between 0·001 and 0·01 nmol/l, and a maximum effective concentration of 1·0 nmol/l. A less steep AVP–ACTH dose-response curve was obtained with a minimum effective concentration of AVP between 0·5 and 5 nmol/l, and no plateau in response up to 5000 nmol AVP/l. Increasing the incubation time between cell preparation and stimulation with AVP from 18 h to 90 h significantly (P <0·01) increased the ACTH response. Repeated stimulation by AVP (100 nmol/l) or CRH (0·01 nmol/l) (5-min pulses every 30 min for 23 pulses) produced ACTH responses which decreased in an approximately exponential curve with time.

When AVP and CRH were given at physiological concentrations, pulse lengths and pulse frequency, the ACTH response to repeated 1-min pulses of AVP, measured as height above basal secretion, was potentiated by the addition of CRH (1, 2·5, 5, 10 and 20 pmol/l) as a constant perifusion at all AVP concentrations tested (1 nmol AVP/l, P < 0·02; 10 nmol AVP/l, P <0·0005; 25 nmol AVP/l, P <0·0005). During the 1-min AVP pulse, the AVP concentration at the level of the cells was 30% of the expected concentration. Potentiation was increased both by increasing AVP concentration (P <0·00005) and by increasing CRH concentration (P <0·00005) up to 5 pmol CRH/l. The ACTH height response to repeated AVP stimulation significantly (P = 0·0034) decreased with time, independent of CRH and AVP concentration. There was a significant (P = 0·014) decrease in ACTH response to CRH infusion with time, independent of CRH concentration.

We conclude that the responsiveness of pituitary cells is markedly influenced by the preparative techniques. The collagenase-dispersed cells, in the in-vitro perifusion system developed, responded to secretagogues which were given at physiological concentrations, pulse lengths and periods. The system thus fulfills our requirements of in-vitro responses reflecting those observed in vivo, and can therefore be used to investigate the multifactorial control of ACTH secretion further.

Journal of Endocrinology (1993) 137, 391–401

Restricted access
S. L. Alexander
Search for other papers by S. L. Alexander in
Google Scholar
PubMed
Close
,
C. H. G. Irvine
Search for other papers by C. H. G. Irvine in
Google Scholar
PubMed
Close
,
J. H. Livesey
Search for other papers by J. H. Livesey in
Google Scholar
PubMed
Close
, and
R. A. Donald
Search for other papers by R. A. Donald in
Google Scholar
PubMed
Close

ABSTRACT

A non-surgical, non-stressful technique was used for collection of pituitary venous blood from five conscious horses every minute for two 10-min periods before and during isolation from the herd, which caused a predictable, yet humane and physiological, emotional stress. Pituitary blood was also sampled every 5 min for two approximately 90-min periods before and after isolation, while jugular blood was sampled every 15 min throughout the experiment.

During isolation, all horses became agitated, hyperventilating and sweating. Packed red cell volume increased, as did pituitary venous concentrations of adrenaline (mean ± s.e.m. concentration before isolation, 621·5±112·3 pmol/l; peak during isolation, 2665·4 ± 869·8 pmol/l; P <0·05) and noradrenaline (before, 871·8 ± 111·8 pmol/l; peak, 2726·1 ± 547·4 pmol/l; P<0·02). Concentrations of arginine vasopressin (AVP) were higher in pituitary venous but not in jugular blood during isolation than during the preceding 10-min period (P <0·05). Although AVP secretion increased in all horses, in three of the five it rose dramatically in the first minute of isolation to 25·7 (horse 1), 13·6 (horse 4) and 145·1 (horse 5) times the level in the last sample collected before isolation. Mean pituitary venous concentrations of ACTH and α-MSH increased during isolation in the three horses which had large increases in AVP secretion, but, overall, stress did not significantly affect ACTH or α-MSH secretion. Similarly, mean jugular cortisol levels were not significantly altered by isolation. However, the magnitudes of ACTH, AVP and α-MSH responses to isolation were negatively correlated with the jugular cortisol level before isolation. The changes in pituitary venous concentrations of ACTH and AVP were synchronous under resting conditions, whether samples were collected at intervals of 1 (P <0·01) or 5 (P <0·005) min; however, this synchrony was lost during isolation. The changes in pituitary venous concentrations of ACTH and α-MSH were synchronous both at rest (P <0·025 for 1-min sampling, P <0·01 for 5-min sampling) and during isolation (P<0·01).

We conclude that isolation stress increases AVP secretion and may alter the temporal relationship between pituitary venous concentrations of AVP and ACTH. Furthermore, the magnitude of the responses of AVP, ACTH and α-MSH to isolation is significantly affected by the prevailing cortisol level.

J. Endocr. (1988) 116, 325–334

Restricted access
M. J. Evans
Search for other papers by M. J. Evans in
Google Scholar
PubMed
Close
,
N. E. Kitson
Search for other papers by N. E. Kitson in
Google Scholar
PubMed
Close
,
J. H. Livesey
Search for other papers by J. H. Livesey in
Google Scholar
PubMed
Close
, and
R. A. Donald
Search for other papers by R. A. Donald in
Google Scholar
PubMed
Close

ABSTRACT

Perifused equine anterior pituitary cells were used to investigate the effect of cortisol on the ACTH response to pulses of corticotrophin-releasing hormone (CRH; 0·01 nmol/l) and arginine vasopressin (AVP; 100 nmol/l), given for 5 min every 30 min for 690 min and ACTH measured in 5-min fractions. At the fourth pulse of secretagogue (0 min), a constant perifusion with cortisol began (0 nmol/l (control), 100, 200, 500, 5000 and 50 000 nmol/l) and continued until the ninth pulse (150 min). For each pulse of secretagogue, the amount of ACTH (pmol) secreted in response to each pulse (ACTH response area), the highest concentration of ACTH (μg/l) measured after each pulse (peak height) and the mean ACTH in the three prepulse fractions (ACTH baseline) were determined. Data from control columns in each experiment were fitted by least squares to an exponential function to produce a mean control value for each end-point; results in all columns were expressed as a percentage of the mean control values.

The addition of cortisol had a highly significant negative effect on ACTH response area, peak height and baseline at all times from + 30 to + 240 min (columns given cortisol compared with the mean of control column values by t-test). Analysis of variance of the data showed that the higher the cortisol concentration, the quicker the ACTH response area (P = 0·0072) and peak height (P = 0·002) decreased to < 50% of mean control, and the greater the maximum percentage change (suppression) in ACTH response area (P <0·0001) and peak height (P <0·0001). The maximum percentage change (suppression) in base-line was independent of cortisol concentration.

At + 30 min after the start of cortisol perifusion, the ACTH response area in CRH columns was significantly lower than in AVP columns (P = 0·0088), and remained lower 90 min after the end of perifusion (P = 0·0084) but the maximum percentage change (suppression) was not different between secretagogues. ACTH peak height was significantly (P < 0·0268) lower in CRH than in AVP columns (from + 30 min until 180 min after the end of cortisol perifusion) and the maximum percentage change (suppression) was also greater (P = 0·0011) in CRH columns.

This study shows the effect of different concentrations of cortisol on CRH- and AVP-induced ACTH secretion by equine anterior pituitary cells, and the time-course for ACTH responses to be inhibited by, and recover from, cortisol perifusion. The highly significant inhibitory effect of cortisol on stimulated ACTH secretion was more apparent when CRH was the secretagogue than when AVP was the secretagogue. The significant inhibitory effect of cortisol on unstimulated baseline secretion of ACTH has not been described previously. These effects occur at physiological concentrations of secretagogues and cortisol. This suggests that, in vivo, circulating cortisol may have an important role in the control of ACTH secretion at pituitary level.

Journal of Endocrinology (1993) 137, 403–412

Restricted access
J. H. Livesey
Search for other papers by J. H. Livesey in
Google Scholar
PubMed
Close
,
H. K. Roud
Search for other papers by H. K. Roud in
Google Scholar
PubMed
Close
,
M. G. Metcalf
Search for other papers by M. G. Metcalf in
Google Scholar
PubMed
Close
, and
R. A. Donald
Search for other papers by R. A. Donald in
Google Scholar
PubMed
Close

First morning urine samples were collected from both menstruant and post-menopausal women and stored at −25 °C. Immunoreactive FSH disappeared from these samples (t½ = 30 days), ultimately stabilizing at about 20% of the initial value. The loss was more rapid at −20 °C and less rapid at −55 °C and +4°C. Immunoreactive LH was also lost from frozen urine, but more slowly than FSH. The addition of glycerol to urine (0·52 mol/l) stored at −25 °C prevented loss of immunoreactive FSH and LH for at least 105 days.

Restricted access
M. J. Evans
Search for other papers by M. J. Evans in
Google Scholar
PubMed
Close
,
J. T. Brett
Search for other papers by J. T. Brett in
Google Scholar
PubMed
Close
,
R. P. McIntosh
Search for other papers by R. P. McIntosh in
Google Scholar
PubMed
Close
,
J. E. A. McIntosh
Search for other papers by J. E. A. McIntosh in
Google Scholar
PubMed
Close
,
J. L. McLay
Search for other papers by J. L. McLay in
Google Scholar
PubMed
Close
,
J. H. Livesey
Search for other papers by J. H. Livesey in
Google Scholar
PubMed
Close
, and
R. A. Donald
Search for other papers by R. A. Donald in
Google Scholar
PubMed
Close

ABSTRACT

A multi-column perifusion system was used to investigate the dynamics of the dose–response relationships of ACTH release by ovine pituitary cells when stimulated by both corticotrophin-releasing hormone (CRF) and arginine vasopressin (AVP) given alone and in combination. A dose–response relationship was obtained when 10-min pulses were given at 60-min intervals over the range of 0·002–2000 nmol CRF/l and 1–2000 nmol AVP/l, with a minimum effective concentration of 0·02 nmol CRF/l or 1 nmol AVP/l. When AVP was given together with CRF, the expected potentiation of the ACTH response occurred when compared with the summed response of these secretagogues given separately. At the higher concentrations of CRF and AVP used, the ACTH responses to repeated pulses decreased with time during the experiment. The rate of this loss of responsiveness was significantly correlated to the size of the response to the first pulse (for CRF: r = 0·89, P<0·01; for AVP: r = 0·95, P < 0·01), being greatest when the response was potentiated by adding the secretagogues together (for CRF plus AVP: r = 0·95, P <0·01). Reduced availability of receptors or changes in intracellular transduction processes may contribute to this desensitization. Reduced levels of secretable ACTH do not appear to be implicated because desensitization to pulses of one secretagogue did not cause equivalent desensitization to the other. In addition, cells stimulated continuously with submaximal levels of either secretagogue showed desensitization while more ACTH was still available for release to higher levels of stimulant. The potentiation of CRF plus AVP resulted primarily from a rapid increase in the height of the response since the width of the response (at one-third maximum height) was significantly (P < 0·001) less after AVP plus CRF than after CRF alone. Also the rapidity with which ACTH concentrations fell after removal of the stimulus from the perifusion medium was significantly (P <0·01) faster following AVP, and CRF plus AVP (P <0·05), than after CRF alone.

It is concluded that reduced ACTH responsiveness following repeated stimulation is dependent upon the type and concentration of the secretagogue. AVP had a shorter duration of action than CRF in vitro and potentiated the initial response to CRF rather than prolonging its action. Desensitization to each stimulant appeared to act by a mechanism independent of the other and therefore appeared to occur at or near the receptor level and be unrelated to the availability of ACTH.

J. Endocr. (1988) 117, 387–395

Restricted access
C J Charles
Search for other papers by C J Charles in
Google Scholar
PubMed
Close
,
S J Rogers
Search for other papers by S J Rogers in
Google Scholar
PubMed
Close
,
R A Donald
Search for other papers by R A Donald in
Google Scholar
PubMed
Close
,
H Ikram
Search for other papers by H Ikram in
Google Scholar
PubMed
Close
,
T Prickett
Search for other papers by T Prickett in
Google Scholar
PubMed
Close
, and
A M Richards
Search for other papers by A M Richards in
Google Scholar
PubMed
Close

Although previous studies have described the hypothalamo–pituitary–adrenal (HPA) response to the stress of acute myocardial infarction, it is not possible to study the hormone changes immediately after infarction in humans. Accordingly, we have examined the HPA response to microembolization of coronary arteries in 13 sheep compared with 5 sham control sheep. Plasma vasopressin (AVP; P<0·001), ACTH (P=0·005) and cortisol (P=0·005) were all increased 2 h (first sample time) after embolization. Plasma ACTH and cortisol levels returned to baseline levels by 6 h but plasma AVP levels did not return to baseline levels until more than 12 h after embolization. Plasma corticotrophin-releasing hormone (CRH) showed no significant change in response to embolization. In a subset of six animals which were sampled more frequently, the peak responses for plasma AVP, ACTH and cortisol occurred at 40 min after embolization. The maximum responses in any individual sheep observed at this time point were 744 pmol/l for AVP, 144 pmol/l for ACTH and 492 nmol/l for cortisol. CRH levels tended to increase across the first hour but these changes were not statistically significant. In conclusion, the stress hormone responses to microembolization of the coronary arteries have been defined in an ovine model of myocardial infarction. This model is suitable for studying the effects of novel treatments to reduce the stress of myocardial infarction.

Journal of Endocrinology (1997) 152, 489–493

Restricted access