The effects were studied of varying the frequency, width and amplitude of pulses of gonadotrophin releasing hormone (GnRH) on the release of LH from anterior pituitary cells. Dispersed sheep cells supported in Sephadex were perifused with medium for 10 h and stimulated with different constant pulse patterns of GnRH. The timing of release of LH was measured by radioimmunoassay of the effluent fractions. Pulses of GnRH ranging in duration from 2 min every 8 min to 16 min every 128 min, and in concentration from 1·7 pmol/l to 250 nmol/l were applied to the cells, as well as continuous stimulation. Comparisons of differences between LH release patterns among samples of the same preparation of cells were used to demonstrate the effects of different GnRH stimulatory regimes. It was concluded that (1) the frequency of GnRH stimulation was important to the nature of LH release (periods shorter than about 16 min between pulses reduced LH output and caused faster desensitization of response), (2) the pulse width of GnRH input was important (the rising edge of the pulse produced greater LH output per unit of GnRH input than did continued application of GnRH within a pulse and wider pulses combined with shorter periods reduced LH output) and (3) over a threshold value of 5–10 nmol GnRH/1 pulse amplitude had little further influence on LH output or rate of desensitization in dispersed cells. These findings reinforce the hypothesis that the rising edge of the GnRH pulse is the major stimulant to LH release.
J. E. A. McIntosh and R. P. McIntosh
Our aim was to determine whether release of LH and FSH can be controlled differentially by the characteristics of applied signals of stimulatory gonadotrophin-releasing hormone (GnRH) alone, free of the effects of steroid feedback or other influences from the whole animal. The outputs of both gonadotrophins were significantly correlated (r≈0·90; P<0·0005) when samples of freshly dispersed sheep pituitary cells were perifused in columns for 7 h with medium containing a range of concentrations of GnRH in various patterns of pulses. Hormone released in response to the second, third and fourth pulses from every column was analysed in detail. Dose–response relationships for both LH and FSH were very similar when cells were stimulated with 5–8500 pmol GnRH/1 in 5-min pulses every hour. When GnRH was delivered in pulses at a maximally stimulating level, the outputs of both hormones increased similarly with increasing inter-pulse intervals. Efficiency of stimulation (release of gonadotrophin/unit stimulatory GnRH) decreased (was desensitized) with increasing pulse duration in the same way for both hormones. Thus, varying the dose, interval and duration of GnRH pulses did not alter the proportions of LH and FSH released in the short-term from freshly dissociated cells. However, the same cell preparations released more LH relative to FSH when treated with maximally stimulating levels of GnRH for 3 h in the presence of 10% serum from a sheep in the follicular phase of its ovulatory cycle compared with charcoal-treated serum. Because there was no gonadotrophin synthesis under the conditions used in vitro these results suggest that changes in the LH/FSH ratio seen in whole animals are more likely to result from differential clearance from the circulation, ovarian feedback at the pituitary, differential synthesis in intact tissue or another hormone influencing FSH secretion, rather than from differences in the mechanism of acute release controlled by GnRH.
J. Endocr. (1986) 109, 155–161
R. P. McIntosh and J. E. A. McIntosh
Pulse amplitude and frequency are often used to describe measurements of LH in blood. Such analyses are compatible with models of LH being released from the pituitary in episodes that are controlled by pulses of hypothalamic gonadotrophin-releasing hormone. The amplitudes of these secretory episodes as seen in blood are usually defined as the net heights of peaks above a baseline. As a measure of each pituitary secretory episode, this is valid only if peaks are regularly and widely spaced, making overlap negligible. When episodes are erratic and frequent so that only fractions of peaks have been cleared from the circulation before others follow, nadirs between peaks include output from previous episodes and do not define a physiologically meaningful baseline. Applied to overlapping peaks, such measures of amplitude usually underestimate pituitary secretory episodes and imply a tonic mode of LH secretion in addition to pulsatile release.
Using the additional information of fitted LH clearance coefficients to define the shapes of LH peaks, a simple method based on an episodic mode of release alone is described, for estimating more accurately the relative sizes of secretory episodes as observed in blood, free of the effects of overlapping peaks. Using this analysis we have described the variation in amplitude, interval and clearance rates of LH secretory episodes within and between four normal menstrual cycles of a single individual. Thirteen, 3–6 h blood sampling sessions were performed during early follicular growth at the transition from luteal to follicular phases when the frequencies of LH peaks, LH/FSH ratios and progesterone concentrations were changing markedly.
Our secretory episode model described all data well without the need to introduce a tonic mode of release. When frequent pulses overlapped we found that amplitudes of episodes were usually higher than peaks estimated by conventional methods, but a decrease in both amplitude and pulse interval occurred after the start of menstruation. Highly variable patterns of LH release were demonstrated in the late luteal phase of this normal individual while FSH levels rose consistently.
J. Endocr. (1985) 107, 231–239
R. P. McIntosh, J. E. A. McIntosh and L. Lazarus
Patterns of hypothalamic stimulation causing pituitary hormone release cannot be studied directly in humans; one possible approach is to make inferences from the nature of the response of the target organ as revealed by patterns of pituitary hormones in blood. Replicated, precise assay of LH in frequently sampled blood of women at differing stages of the menstrual cycle has demonstrated previously that secretion of this hormone is compatible with a model of discrete, instantaneous episodes of LH output, which are assumed to be stimulated by isolated bursts of increased stimulatory hypothalamic gonadotrophin-releasing hormone. However, similarly detailed measurements of the dynamic secretion patterns of GH in women reported here, revealed much slower rates of increase of GH concentrations (median time to maximum concentration 38 min) in comparison with LH (13 min) assayed in the same blood samples. These rise rates of GH were uncorrelated with the final amplitude of the peak and were observably discontinuous in half the peaks. Simultaneous i.v. injection of a bolus of mixed GRF and GnRH produced similar dynamics of pituitary release of GH and LH. Thus differences in patterns of natural release of the two hormones appear to be contributed to by differences in the modes of hypothalamic stimulation. Current understanding of control of GH release in animal models suggests that the slow-rising, frequently discontinuous natural peaks of GH in human blood are likely to be caused by interaction between the withdrawal of inhibitory hypothalamic somatostatin and the increased secretion of stimulatory GRF.
J. Endocr. (1988) 118, 339-345
R. P. McIntosh, J. E. A. McIntosh and L. Starling
This study investigated the importance of reorganization of cell components by cytoskeletal structures to the short-term dynamic changes in LH release from dispersed sheep pituitary cells in perifusion, when stimulated with different dynamic patterns of gonadotrophin-releasing hormone (GnRH). The changes in rate of LH release investigated were the initial response to GnRH, desensitization, change of dose–response during desensitization, and recovery of sensitivity between pulses of stimulation. Cytochalasin D and colchicine were used to modify microfilament and microtubule action respectively. To determine whether receptor movement after binding of agonist was involved in the altered responses, K+ and phorbol 12-myristate 13-acetate (PMA) were used as stimulants because they cause LH release independently of agonist-receptor interaction.
After 3 and 48 h culture on dextran beads and 2–3 h incubation in the presence and absence of 2–48 μmol cytochalasin D/l, or 8 or 250 μmol colchicine/l, aliquots of collagenase-dispersed sheep pituitary cells were stimulated at 37 °C in tubes or in a multicolumn perifusion system with 850 pmol GnRH/l, 109 mmol K+/l or 10 nmol PMA/l. Fractions of supernatant or effluent were collected at intervals and LH concentrations measured by radioimmunoassay. Control samples were treated in the same way but without stimulation.
Maximal, reversible enhancement of LH release over the first 20 min following stimulation with all secretogogues was observed after incubation of cells in 6 μmol cytochalasin/l. Desensitization behaviour, the supramaximal response, and the ability of cells to recover sensitivity to repeated pulses of GnRH were not altered by this modifier of microfilament polymerization at 6 or 24 μmol/ml. Colchicine at 8 μmol/l caused no changes in LH release. At 250 μmol/l, colchicine reduced the initial response of cells to GnRH stimulation but its action at this relatively high level may not be specific; there was no other major change in desensitization patterns, nor recovery of sensitivity to pulsed GnRH stimulation. Each treatment affected cellular responses similarly before and after culture.
From studying the details of the dynamics of the short-term responses of gonadotrophs, we conclude that transport of cell components involving microfilaments and microtubules is unlikely to be a major limitation on the rate of LH release during desensitization, the supramaximal response, or the recovery of sensitivity between pulses of GnRH. This suggests that biochemical reactions rather than physical translocation may be rate-limiting in these processes. In addition, although inhibition of microfilament action does appear to enhance the earliest observed response to stimulation of the LH-release mechanism, this occurs after protein kinase C activation and is probably not related to impairment of processes such as polymerization and sequestration of agonist-bound GnRH receptors because the effects are also observed with K+ and PMA, stimulants acting independently of agonist-receptor interaction.
J. Endocr. (1987) 112, 289–298
L. Starling, J. E. A. McIntosh and R. P. McIntosh
We report an estimate of the rate of externalization of unstimulated receptors for gonadotrophin-releasing hormone (GnRH), and derive from this the turnover time of the unstimulated receptor. The binding of the GnRH antagonist acetyl-d-pCl-Phe1,2,d-Trp3,d-Lys6,d-Ala10]-GnRH to dispersed sheep anterior pituitary cells was non-saturable at 37 °C. Further experiments showed that the binding had two distinct phases. We suggest that these phases correspond to the initial, saturable binding to existing plasma membrane receptors, followed by binding to receptors as they are inserted into the surface membrane. The two processes are temporally distinct, and can be inhibited independently by pharmacological manipulations. The initial phase was inhibited by treatments that could be expected to reduce the number of active receptors on the cell surface (preincubation of the cells for 30 min with 100 μg neuraminidase/ml or 50 μmol GnRH/ml), and was complete in less than 30 min after the addition of the antagonist tracer. The second phase occurred continuously in the presence of tracer, and was reduced or abolished by inhibitors of microtubule function (100 μmol vinblastine/l), protein synthesis (25 μg cycloheximide/ml), or energy metabolism (0·25 mmol 2,4-dinitrophenol/l). The rate of insertion of receptors into the plasma membrane was calculated from the rate of increase of the second phase of binding. The calculated rate implies a 100% turnover of unstimulated receptors every 150 min. In contrast, previously published estimates of the rate of internalization of the GnRH–receptor complex in the rat pituitary suggest that the stimulated receptor is turned over much faster.
J. Endocr. (1988) 117, 97–107
L. Starling, R. P. McIntosh and E. A. Mclntosh
The possible involvement of polyphosphoinositides in the stimulation of LH release was investigated. Dispersed sheep pituitary cells were incubated in test-tubes, or perifusedns in columns, with gonadotrophin-releasing hormone (GnRH) and Li+, or with a phorbol ester, and the amounts and patterns of LH release over time compared.
Treatment with Li+ (10 mmol/l), which is known to increase levels of inositol phosphates in gonadotrophs, was shown to have effects only on the responses of desensitized cells, significantly decreasing the rate at which the cells desensitize (P<0·005) and decreasing the response to supramaximal levels of GnRH stimulus (P<0·01). It is suggested that these effects could be due to increased levels of inositol monophosphate, inositol bisphosphate inositol 1,3,4-trisphosphate. Responses to single or repeated pulses of GnRH at 18-, 30- and 60-min intervals were not significantly altered.
Phorbol 12-myristate 13-acetate (PMA), an activator of the calcium and phospholipid-dependent protein kinase (protein kinase C), was specifically active in releasing LH with a half-maximal stimulating dose of approximately 3 nmol/l. Phorbol 12,13-diacetate, which is structurally similar to PMA but does not activate protein kinase C, did not release LH, except at high levels in freshly dispersed cells. The timing of PMA-stimulated LH release was similar to that for GnRH-stimulated release, and PMA was able to release greater amounts of LH than could GnRH. This suggests that activation of protein kinase C is likely to be important in the GnRH-stimulated release of LH from gonadotrophs. It also shows that the desensitization to GnRH stimulation observed after 10 min is unlikely to be caused by lack of releasable LH. Cells desensitized to maximally stimulating levels of GnRH still responded strongly to PMA stimulation, indicating that the desensitization to GnRH stimulation involves a step in the transduction mechanism before activation of protein kinase C.
J. Endocr. (1986) 111, 167–173
M. J. Evans, J. T. Brett, R. P. McIntosh, J. E. A. McIntosh, J. L. McLay, J. H. Livesey and R. A. Donald
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