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J E McIntosh

Publications and source records attributed to J E McIntosh.

At least 19 recordsLinked to original sources
PubMed2003

Meal pattern changes associated with temporomandibular joint inflammation/pain in rats; analgesic effects.

Establishing a valid animal model to study temporomandibular joint (TMJ) pain has proven extremely difficult. Using complete Freund's adjuvant (CFA) to induce TMJ inflammation, we recently showed that meal pattern analysis could be used as a noninvasive biological marker to study TMJ pain in an animal model. The purpose of this study was to further validate our animal model by determining whether aspects of CFA-induced TMJ inflammation/pain are reversed with ibuprofen (IBU) treatment. In the first trial, 48 male rats were used and in the second trial, 32 female ovariectomized rats, given 17beta-estradiol replacement, were used. The rats were assigned to one of four groups: control (CON-CON); control+IBU (CON+IBU); CFA-CON; and CFA+IBU. In the male trial, CFA injection (P<.01) caused TMJ swelling and chromodacryorrhea (CFA-CON); IBU eliminated these changes in the CFA+IBU group. Meal pattern analysis showed the pertinent CFA-induced change and the IBU effect was that meal duration was increased in the CFA-CON group (P<.01), but normal in the CFA+IBU-treated group on the first, but not second, day postinjection. In the female trial, CFA increased TMJ swelling, but did not cause significant chromodacryorrhea (CFA-CON); IBU eliminated swelling in the CFA+IBU group. Meal duration was increased (P<.01) in the CFA-CON group, but was normal in the CFA+IBU-treated group on both the first and second days postinjection. In both trials, interleukin-1beta (IL-1beta) levels were increased similarly in CFA-CON and CFA+IBU groups (P<.01). This study shows that CFA-induced TMJ inflammation/pain can cause changes in meal patterns (i.e., meal duration), which may be used as a behavioral marker for TMJ inflammation/pain.

Animals↗
PubMed2001

Modulation of the inflammatory response in the rat TMJ with increasing doses of complete Freund's adjuvant.

OBJECTIVES: Acute inflammation stresses the physiological system, which must respond in order to reestablish homeostasis. The purpose of this study was to determine whether bilateral temporomandibular joint (TMJ) injections of different doses of Complete Freund's Adjuvant (CFA) produced dose-dependent changes in biologic markers of acute inflammation. The ability to establish an animal model with varying degrees of joint inflammation would allow evaluation of agents or conditions that could modulate the severity of the disease. DESIGN: The TMJs of three groups of male Sprague-Dawley rats were injected with CFA containing varying doses of Mycobacterium tuberculosis (MT). A group of non-injected and a group of saline injected rats were used as controls. Food intake, body weights, swelling and chromodacryorrhea were recorded daily. Interleukin-1 beta (IL-1 beta) and corticosterone levels were assayed and condylar cartilage thickness was measured 48 h after injections. RESULTS: Twenty-four hours post-injection, bilateral TMJ swelling and chromodacryorrhea were significantly (P< 0.05) increased following 10 microg of MT and further increased with elevated MT dose. In the CFA groups food intake was attenuated (P< 0.01) 24 and 48 h post-injection and negatively correlated with dose at 24 h. Body weight was also negatively correlated with dose. TMJ retrodiscal tissues IL-1 beta was increased (P< 0.05) in a dose-dependent manner. CFA increased corticosterone (P< 0.05), but this elevation was not dose dependent. Condylar cartilage thickness was decreased in a dose-dependent manner. CONCLUSIONS: These data suggest that an intermediate dose of CFA can be used to effect submaximal levels of TMJ inflammation that will allow experimental modulation in future studies.

Acute-Phase Reaction↗
PubMed2000

Meal pattern analysis in response to temporomandibular joint inflammation in the rat.

Inflammation of the temporomandibular joint (TMJ) can alter behavioral responses such as food intake and mobilize stress hormones. The hypothesis of this study was that food intake and diurnal corticosterone analysis can be used as indicators of adjuvant-induced TMJ inflammation. Groups of rats received adjuvant or no injections at the beginning of the resting (AM) or activity (PM) phase. Forty-eight hours (early) or 6 weeks (late) after adjuvant injection, plasma corticosterone was assayed and food intake was recorded. Food intake was suppressed up to 4 days post-injection. As expected, the non-injected group showed low AM and high PM corticosterone. AM corticosterone was elevated, but PM corticosterone was attenuated in both early- and late-stage-injected rats. A computerized pair-fed experiment showed that adjuvant-induced hypophagia did not alter corticosterone levels. Meal pattern analysis revealed decreased food intake due to a decrease in the number of meals taken. Notably, meal size remained the same but meal duration increased. This model demonstrated that food intake and stress hormone analysis could be used as indicators for sequelae of adjuvant-induced TMJ inflammation.

Analysis of Variance↗
PubMed1996

Effect of cortisol on percentage of non-sex-hormone-bound steroid: implications for distribution of steroids on binding proteins in serum.

We previously observed that in men concentrations of serum testosterone (T) not bound to sex-hormone-binding globulin (n-SHBGT) decreased as concentrations of cortisol increased in early morning. This led us to investigate in vitro the influence of several steroids on protein-bound T. Steroids were added to late-evening sera containing low concentrations of cortisol. Changes were measured in percent T or estradiol not bound to SHBG (%n-SHBGT or %n-SHBGE). Results were compared with computer simulations of a mass action model describing current understanding of steroid binding to serum proteins. In vitro measurements confirmed changes observed in vivo. Cortisol at 600 nmol/L reduced %n-SHBGT to 61% +/- 5% of basal, but this was reversed with cortisol at 2000 nmol/L. Progesterone reduced %n-SHGBT less, and dexamethasone had no effect. Free T rose with added cortisol. Increasing estradiol to 900 nmol/L caused an increase in %n-SHBGT. The %n-SHBGE rose with added cortisol (121% +/- 5% of basal with cortisol at 1000 nmol/L). Simulation predicted all behaviors except the marked initial decrease in %n-SHBGT as cortisol concentrations increased and the absolute values of %n-SHBGT and %n-SHBGE. A possible explanation for the former is that T is displaced from corticosteroid-binding globulin (CBG) by added cortisol, more T is bound to CBG than expected, and T displaced from CBG associates with SHBG rather than albumin. Alternatively, current understanding about steroid binding to serum proteins has other major deficiencies.

Binding, Competitive↗
PubMed1995

The influence on a 'normal' birthweight distribution of a minor population of growth retarded infants: a Monte Carlo simulation.

It is common practice to define babies below a certain birthweight as 'growth-retarded' or 'small-for-dates'. This presupposes that there is a specific subpopulation of babies who have not achieved their genetic growth potential. The presence of this subpopulation should result in a deviation from a normal distribution in the population as a whole. We have used a mathematical simulation, together with data from an actual sample of 1463 male Caucasian neonates delivered at 40 weeks, in order to define the characteristics of such a subpopulation which would yield deviations from the normal sufficient to be statistically significant. On the basis of this study, there was no evidence for such a subpopulation of low birthweight babies in the 1463 neonates observed.

Birth Weight↗
PubMed1993

Circadian variation in serum free and non-SHBG-bound testosterone in normal men: measurements, and simulation using a mass action model.

OBJECTIVE: We measured the changes in testosterone fractions in serum of normal men over a 24-hour period, and determined whether they could be simulated on the basis of current understanding of the interactions between steroids and binding proteins in the blood. DESIGN: Starting from between 0830 and 0930 h, blood samples were taken every 45 minutes for 25.5 hours. PATIENTS: Five healthy males aged 26-45 years. All participants worked on a hospital campus and while being sampled carried out their normal activities during waking hours. MEASUREMENTS: The concentrations of testosterone (RIA) and albumin, and the percentage non-sex hormone binding globulin-bound testosterone (ammonium sulphate precipitation) and percentage free testosterone (rate dialysis), were measured on each sample. Cortisol (RIA) and sex hormone-binding globulin (SHBG) (IRMA) concentrations were measured on every second sample, and that of corticosteroid-binding globulin on two samples from each series. RESULTS: In all participants the levels of free and non-SHBG-bound testosterone in early morning samples (near 0530 h) were significantly different from those taken before midnight (P < 0.0005). Significant circadian rhythms (P < 0.05) in the concentration of testosterone and in the level of the free fraction were detected in all participants, and in four of the five participants for the non-SHBG-bound fraction. The amplitude of the free testosterone rhythm (34 +/- 2% of basal) was greater than that for testosterone itself (24 +/- 3% of basal). The 24-hour rhythm of the non-SHBG-bound fraction was similar to the total and free fractions except for the period 0330-0900 h when the level of this fraction declined by 15-45% over 1.5-3 hours. This decline was coincident with the initial rise in the concentration of cortisol. A decline of 10.5 +/- SEM 1.0% in the concentration of albumin, and 12.0 +/- 1.1% in that of SHBG occurred when the mean ambulant and supine levels were compared; analysis indicated significant circadian rhythms in the concentrations of these proteins. Simulation was used to investigate possible causes for the circadian rhythms in free and non-SHBG-bound testosterone. Simulation results matched the measured data well in qualitative terms, but quantitatively there were differences. CONCLUSIONS: Increasing saturation of the binding proteins following rises in testosterone production, and the small but significant changes in protein concentration, probably related to postural changes, were implicated as the major factors in the rhythm amplitude. However, the early morning decline in the non-SHBG-bound fraction was not explained by these factors. The rise in cortisol concentration at this time is a probable cause. Alternatively, simulation suggests that a substance appearing in the early morning and competing with testosterone for albumin binding sites may be responsible.

Adult↗
PubMed1992

Angiotensin II and acetylcholine differentially activate mobilization of inositol phosphates in Xenopus laevis ovarian follicles.

Angiotensin II (AII) evokes a Ca(2+)-dependent Cl- current in Xenopus laevis ovarian follicles that appears to involve a pertussis-toxin-sensitive G protein mediating phosphoinositide hydrolysis and Ca2+ mobilization from intracellular stores. Follicle responses to AII closely resemble the two-component response stimulated by acetylcholine (ACh) in this tissue. Intraoocyte injections of phytic acid, heparin, and inositol 1,4,5-trisphosphate [Ins(1,4,5)P3], acting as inhibitors of Ins(1,4,5)P3-induced Ca(2+)-release, resulted in loss of responsiveness to AII and ACh. As previously reported for ACh [Moriarty et al. (1988) Proc Natl Acad Sci USA 85: 8865-8869], pertussis toxin and microinjected GTP[gammaS] were found to inhibit follicle responses to AII, implying the involvement of a G protein. However, ACh and AII responses differ strikingly in the way they mobilize inositol phosphates and in densitization characteristics. We have previously been unable to find significant increases in inositol phosphates after 60 min stimulation (with Li+) by AII, although ACh potently activated increases in these [McIntosh and McIntosh (1990) Arch Biochem Biophys 283: 135-140]. In the present paper, AII was found to activate rapid increases in inositol bis- and trisphosphates after 1 min stimulation without Li+. ACh and AII also exerted different actions on follicle adenylate-cyclase-dependent responses. We conclude that at least two separate inositol-phosphate-linked receptor mechanisms may exist in ovarian follicles, resulting from involvement of one or more pertussis-toxin-sensitive G protein(s).

Acetylcholine↗
PubMed1991

Mathematical analysis of perifusion data: models predicting elution concentration.

System models are constructed and analyzed for combined convective flow and for dispersion in distorting concentrations of a chemical signal as it passes through a packed column. We derive general analytical solutions for these models. The results have applications to analyses such as in biological experiments involving hormonal stimulation of perifused cells, elution chromatography, adsorption columns, and studies of groundwater flow. The models reveal that the column distorts an incoming signal (such as a change in solute concentration in the flowing liquid) at the inlet. This distortion is greatest at low values of the Peclet number of the flow and is small at larger values. We explore the effects of the approximations inherent in the mathematical models of the system. Specification of the boundary conditions of the problem are shown to be particularly important. With the use of incorrect models, it is possible to obtain accurate interpolations to data obtained from perfusion experiments. However, the parameters derived (in particular the dispersion constant and the peak concentration of a solute concentration pulse) may be considerably in error. This may lead to errors when these parameter estimates are used to predict results in other experimental situations.

Animals↗
PubMed1990

Diversity in responses from endogenous and expressed mammalian receptors which cause chloride ion efflux from ovarian follicles of Xenopus laevis.

Inositol phosphates are produced in ovarian follicles of Xenopus laevis on activation of endogenous acetylcholine receptors, which also stimulates Ca2+ release and efflux of Cl- ions detected electrophysiologically. Inositol phosphates were not detectable on activation of endogenous angiotensin II receptors which did, however, stimulate both a dose-dependent Ca2+ efflux and a depolarizing current very similar in maximum size and other characteristics to those caused by acetylcholine action. In contrast, activation of exogenous receptors for angiotensin II expressed by microinjected mRNA extracted from bovine adrenal did form measurable inositol phosphates. Also, the endogenous electrophysiological responses to angiotensin II and acetylcholine desensitize homologously but fail to cross-desensitize (Lacy, McIntosh, and McIntosh, 1989, Biochem. Biophys. Res. Commun, 159, 658-663). It appears that endogenous ovarian angiotensin II receptors in Xenopus activate a different transduction mechanism from endogenous acetylcholine receptors and expressed mammalian adrenal angiotensin II receptors and/or may be sited in the electrically connected follicular cells rather than in the oocyte itself.

Acetylcholine↗
PubMed1990

Metabolism of the biologically active inositol phosphates Ins(1,4,5)P3 and Ins(1,3,4,5)P4 by ovarian follicles of Xenopus laevis.

The metabolism of biologically active inositol phosphates in developed ovarian follicles from Xenopus laevis was investigated. Techniques used were microinjection of tracer into the intact oocyte coupled by gap junctions to follicle cells, as well as addition of tracer to homogenates of ovarian follicles and to homogenates of oocytes stripped of outer follicle-cell layers. Metabolism was similar to that previously described for other types of cell and tissue, with several unusual features. Homogenates of ovarian follicles were shown to contain an apparent 3'-phosphomonoesterase capable of converting [3H]Ins(1,3,4,5)P4 predominantly into a substance with h.p.l.c. elution characteristics of Ins(1,4,5)P3. In intact ovarian follicles, little Ins(1,4,5)P3 was formed but the esterase was activated by the phorbol ester activator of protein kinase C, PMA (phorbol 12-myristate 13-acetate; 60 nM), as well as by acetylcholine (200 microM). In follicle homogenates, this enzyme also appeared to be active in converting [3H]Ins(1,3,4)P3 into a substance eluting as Ins(1,4)P2. The apparent 3'-phosphomonoesterase activity was not inhibited by intracellular (or higher) levels of Mg2+. Although PMA activated this enzyme in intact oocytes relative to 5'-phosphomonoesterase activation, it did not enhance overall metabolism, in contrast with reports on other tissues. Compared with the processing of inositol phosphates injected into the intact follicle, homogenization in simulated intracellular medium appeared to alter the activity and/or accessibility of several enzymes. The metabolism of inositol phosphates appears to occur predominantly in the follicle cells surrounding the oocyte, as collagenase treatment followed by defolliculation greatly diminished the rates of metabolism of several inositol phosphates. The presence in Xenopus ovarian follicles of a 3'-phosphomonoesterase activated by protein kinase C in addition to the well-known 3'-kinase suggests that, by forming a reversible interconversion between Ins(1,4,5)P3 and Ins(1,3,4,5)P4, this tissue may have the potential to prolong stimulatory signals on binding of appropriate agonists to receptors.

Acetylcholine↗
PubMed1990

Formation of inositol pentakisphosphate by ovarian follicles of Xenopus laevis from metabolism of inositol (1,4,5)trisphosphate and inositol (1,3,4,5)tetrakisphosphate and from receptor activation.

Small amounts of a higher inositol phosphate with chromatographic properties of [3H]inositol (1,3,4,5,6)pentakisphosphate were formed from [3H]inositol (1,4,5)trisphosphate added to homogenates of ovarian follicles of Xenopus laevis, and from [3H]inositol (1,3,4,5)tetrakisphosphate after injection into follicular oocytes. Other intermediate forms of inositol tetrakisphosphate were not detectable. [3H]inositol (1,3,4,5,6)pentakisphosphate prepared from chicken erythrocytes was metabolized in homogenates to an inositol tetrakisphosphate eluting later than the (1,3,4,5) isomer. Activation of receptors in ovarian follicles of Xenopus laevis with acetylcholine or stimulation with injected GTP gamma S caused formation not only of inositol trisphosphate and its expected metabolites but also of small amounts of inositol pentakisphosphate. These results suggest that the latter may be formed from metabolites of inositol (1,4,5)trisphosphate in this tissue during receptor activation.

Acetylcholine↗
PubMed1989

Angiotensin II stimulates an endogenous response in Xenopus laevis ovarian follicles.

While responses to angiotensin II have previously been induced in Xenopus laevis oocytes after injection of messenger RNA extracted from mammalian tissue, no endogenous responses of ovarian tissue to this hormone have been reported. Here we describe such an endogenous dose-dependent response to angiotensin II, detected by conventional electrophysiological techniques, in follicular oocytes. The ED50 of the response was estimated to be 0.15 +/- 0.07 microM (S.E.M.). Maximal depolarization, obtained at 1 microM angiotensin II, was 18.3 +/- 1.4 mV (n = 18, three experiments using oocytes from two toads, mean resting membrane potential = -42 +/- 2 mV). The response was absent from collagenase-treated oocytes or follicular oocytes treated with octanol, suggesting that the receptors are predominantly in the follicular layer surrounding the oocytes.

Angiotensin II↗
PubMed1988

Peptidyl-glycine alpha-amidating mono-oxygenase activity towards a gonadotropin-releasing-hormone C-terminal peptide substrate, in subcellular fractions of sheep brain and pituitary.

The amidation of a synthetic peptide D-Tyr-Pro-Gly-Gly by sheep hypothalamic and pituitary preparations was measured. This substrate was designed as a glycine-extended C-terminal peptide analogue of gonadotropin-releasing hormone (GnRH) to test the ability of these tissues to convert the product produced by cleavage of the GnRH prohormone into the active amidated decapeptide. An alpha-amidating activity capable of converting D-125I-Tyr-Pro-Gly-Gly into D-125I-Try-Pro-Gly-NH2 was identified in crude synaptosomal and neurosecretory-granule fractions from hypothalamus and anterior-pituitary secretory-granule preparations. This activity was stimulated by the addition of Cu2+ and reduced ascorbate, and was maximal at neutral pH in sulphonic acid buffers. Highest activity was measured in synaptosomes from the median eminence and medial basal hypothalamus and in pituitary granules. Lower activity was found in synaptosomes prepared from anterior hypothalamic tissue. Negligible activity was measurable in cerebral cortex and none in pineal synaptosomes. Direct comparison of alpha-amidation with D-125I-Try-Pro-Gly-Gly and a previously reported substrate D-125I-Tyr-Val-Gly showed that, although the latter was 15-20-fold more reactive, the optimal concentration of Cu2+ for amidation was similar with both substrates in medial-basal-hypothalamic synaptosomes and pituitary granules. Activity measured with 1 microM-D-125I-Tyr-Val-Gly was inhibited by increasing concentrations of D-Tyr-Pro-Gly-Gly, with 50% inhibition at 25 microM-D-Tyr-Pro-Gly-Gly, whereas activity with 3.3 microM-D-125I-Tyr-Pro-Gly-Gly was abolished by addition of 1 microM-D-Tyr-Val-Gly, evidence that the two substrates were competing for the same enzyme activity. Synaptosomal preparations demonstrated Michaelis-Menten kinetics for D-Tyr-Pro-Gly-Gly as substrate, with values of Km and V decreasing upon removal of ascorbate. We conclude that D-Tyr-Pro-Gly-Gly-directed alpha-amidation in sheep hypothalamic synaptosomes resembles the activity with D-Tyr-Val-Gly as substrate, as well as that demonstrated by others with D-Tyr-Val-Gly as substrate in rat hypothalamic and pituitary tissue. Although reactivity towards D-Tyr-Pro-Gly-Gly cannot be assumed to assess amidation solely of GnRH, the negligible D-Tyr-Pro-Gly-Gly-directed activity in the pineal gland and cerebral cortex, areas that are known to synthesize other alpha-amidated peptides, suggests some substrate specificity in alpha-amidating enzymes from different tissues.

Animals↗
PubMed1988

Estimating the rate of externalization of gonadotrophin-releasing hormone receptors in ovine anterior pituitary cells in vitro.

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 degrees 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 micrograms neuraminidase/ml or 50 mumol 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 mumol vinblastine/l), protein synthesis (25 micrograms 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.

Animals↗
PubMed1988

Characteristics of the ACTH response to repeated pulses of corticotrophin-releasing factor and arginine vasopressin in vitro.

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/1 and 1-2000 nmol AVP/1, with a minimum effective concentration of 0.02 nmol CRF/1 or 1 nmol AVP/1. 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 less than 0.01; for AVP: r = 0.95, P less than 0.01), being greatest when the response was potentiated by adding the secretagogues together (for CRF plus AVP: r = 0.95, P less than 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.(ABSTRACT TRUNCATED AT 250 WORDS)

Adrenocorticotropic Hormone↗
PubMed1988

A comparison of the dynamics of secretion of human growth hormone and LH pulses.

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.

Female↗
PubMed1987

Effects of modifiers of cytoskeletal structures on the dynamics of release of LH from sheep anterior pituitary cells stimulated with gonadotrophin-releasing hormone, K+ or phorbol ester.

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 mumol cytochalasin D/1, or 8 or 250 mumol colchicine/l, aliquots of collagenase-dispersed sheep pituitary cells were stimulated at 37 degrees C in tubes or in a multicolumn perifusion system with 850 pmol GnRH/1, 109 mmol K+/1 or 10 nmol PMA/1. 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 secretagogues was observed after incubation of cells in 6 mumol 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 mumol/ml. Colchicine at 8 mumol/l caused no changes in LH release. At 250 mumol/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.(ABSTRACT TRUNCATED AT 400 WORDS)

Actin Cytoskeleton↗