Continuous subtherapeutic insulin counteracts hypothalamopituitary-gonadal alterations in diabetic rats.

In experimental animal models, gonadal axis lesions are probably responsible for reproductive disorders associated with diabetes mellitus. The pathogenesis of these disorders is not yet known, but it is assumed that insulin deficiency plays an important role. To check this hypothesis, we have investigated the hypothalamopituitary-gonadal axis of insulin-treated streptozocin-induced diabetic (STZ-D) rats and compared it with that of untreated diabetic and control animals. Insulin was delivered by subcutaneously implanted osmotic minipumps. Furthermore, to determine whether possible beneficial insulin effects are selectively limited to the gonadal axis or act generally, we also studied retinal microangiopathy. The hypothalamopituitary-gonadal axis of insulin-treated diabetic animals was almost unchanged. On the contrary, retinal microangiopathy was only slightly influenced by subtherapeutic insulin doses. In conclusion, continuous administration of insulin at subtherapeutic doses can successfully counteract most of the effects of diabetes on the gonadal axis. Thus, the gonadal-axis impairment in STZ-D animals appears to be related to the fall of plasma insulin below a critical level. Furthermore, the various organ systems may respond to different plasma insulin threshold levels.

Reverse hemolytic plaque assay study of luteinizing and follicle-stimulating hormone and thyrotropin secretion in diabetic rat pituitary glands.

Numerous studies indicate that an impaired hypothalamopituitary axis plays an important role in reproductive and thyroid disorders in diabetic humans and animal models. Yet, several questions about the pathogenesis of these diabetic complications have not been answered. To evaluate the basal secretion of single gonadotrophs and thyrotrophs in vitro, uncultured pituitary cells from control rats and 1-mo streptozocin-induced diabetic (STZ-D) rats were studied with a reverse hemolytic plaque assay and morphometry. After light-microscopy immunocytochemistry for gonadotropin and thyrotropin (TSH), we recorded the ratio of plaque-forming to non-plaque-forming cells. The area of plaques produced by luteinizing hormone (LH), follicle-stimulating hormone (FSH), and TSH cells and the area of plaque-forming and non-plaque-forming cells were clearly smaller in diabetic than control rats. The plaque area, however, was more severely reduced than the cell area. The percentage of LH-, FSH-, and TSH-immunoreactive plaque-forming cells was greatly decreased in diabetic compared with control animals. In conclusion, our findings demonstrate that the LH-, FSH-, and TSH-secreting cells of diabetic rats released less hormone and were less numerous than the corresponding cells of control rats. Thus, several pathogenetic mechanisms might be involved in reduced gonadotropin and TSH release at the cellular level: 1) anatomical lesions of organelles involved in glycoprotein hormone synthesis and secretion, possibly due to insulin deficiency; 2) decreased gonadotropin-releasing hormone (GnRH) and thyrotropin-releasing hormone (TRH) receptors on pituitary cells; 3) inadequate GnRH and TRH stimulation; 4) high plasma corticosterone levels; or 5) a combination of points 1-4.

Functional and morphological changes in mediobasal hypothalamus of streptozocin-induced diabetic rats. In vitro study of LHRH release.

To investigate the role of the mediobasal hypothalamus (MBH) in diabetic gonadal axis disorders, the MBHs of adult male streptozocin-induced diabetic (STZ-D) rats were examined after incubation in basal conditions or in K+-enriched medium and compared with those of controls. Diabetes lasted 1 mo. Both luteinizing-hormone-releasing hormone (LHRH) release and MBH morphology were studied. After incubation in basal conditions, the LHRH release was unchanged. By light microscopy, the dilated-axon cross sections were more numerous (P less than .01) in the basal arcuate nucleus and in the median eminence. By electron microscopy, the ratio of exocytoses to neurosecretory granules observed in the median eminence axon cross sections was smaller (P less than .05). The total LHRH immunoreactivity, the number of labeled axons, and the amount of positive material in the axons were reduced (P less than .05). After incubation in K+-enriched medium, the LHRH release was markedly reduced (P less than .01). The number and area of dilated-axon cross sections, possibly because of the relation between exocytosis and physiological dilation, were less augmented (P less than .01). Whereas the number of exocytoses and the ratio of exocytoses to neurosecretory granules were not decreased, the total LHRH immunoreactivity and the number of labeled axons were reduced (P less than .05). The releasable LHRH pool therefore seems to be exhausted in control MBH because of long-term stimulation and reduced in the MBH of STZ-D rats because of diabetes. In conclusion, STZ-D causes functional and anatomical MBH lesions that should be pathogenetically relevant for the disorders of the gonadal axis documented in this animal model.

Functional and morphological aspects of impaired TRH release by mediobasal hypothalamus of STZ-induced diabetic rats.

Streptozocin-induced diabetes (STZ-D) in rats is associated with marked hypothyroidism characterized by functional impairment and structural lesions of the pituitary-thyroid axis. Degenerative axonal lesions, which can be prevented by insulin administration, have been reported in the mediobasal hypothalamus (MBH) of STZ-D rats. However, direct evidence connecting anatomic MBH lesions with functional impairment is still missing. We therefore performed a combined functional and morphological investigation in 4-mo-old STZ-D male rats (diabetes lasted 1 mo), applying an in vitro model to study in the same isolated MBH 1) the basal and depolarization-induced thyrotropin-releasing hormone (TRH) release during two successive incubations of 20 min each and 2) morphological and morphometric aspects, including distribution and amount (densitometric evaluation) of immunoreactive TRH in the incubated tissue. In basal conditions, TRH release was much lower in diabetic than control MBH during both incubations (P less than .01 vs. P less than .05). In depolarizing conditions, TRH release was increased during the second incubation in control (P less than .05) and during both incubations in diabetic (P less than .01) rats, the percentage increase of the TRH release due to ionic stimulation being much higher in diabetic than control animals (P less than .01). As determined by light-microscope morphometry, the total area of dilated-axon cross sections was larger in diabetic than control MBH under basal conditions (P less than .01), thus confirming degenerative axonopathy in diabetic rats. By densitometry determination, the amount of immunoreactive TRH was higher in stimulated diabetic MBH compared with both stimulated control and basal diabetic MBH (P less than .01).(ABSTRACT TRUNCATED AT 250 WORDS)

Crystallization of an odorant-binding protein from cow nasal mucosa.

The first odorant-binding protein isolated from mammalian nasal mucosa is a dimer of subunits of identical molecular weight (19,000) that specifically binds bell pepper odorants, "green" smelling compounds. The protein can be purified in milligram quantities from tissue extractions by sequential use of a silica based anion exchange column and Mono-P chromatofocussing column. In the presence of the binding compound 2-isobutyl-3-methoxypyrazine and of the organic solvent 2-methyl-2,4-pentanediol (17%, v/v), the protein crystallizes in the monoclinic space group P2(1), with unit cell constants a = 54.3 A, b = 66.7 A, c = 41.5 A, beta = 97.2 degrees. From consideration of the crystal packing densities compatible with its unit cell, it can be concluded that two subunits of 19,000 Mr each are present in the asymmetric unit. The diffraction pattern on still photographs of this crystal form of the protein extends to 2.5 A resolution and allows for a detailed crystallographic investigation.

Structure and oxygen affinity of crystalline desArg141 alpha human hemoglobin A in the T state.

The correlation of a protein structure determined crystallographically to its functional properties determined in solution can be an extremely complex problem due to potential differences of protein conformational flexibility in the two physical states. A more direct approach to the correlation of structure with function is to examine both the structure and the function of a protein in the same crystalline environment. In this paper, the structural and functional properties of T state desArg hemoglobin (human hemoglobin modified by removal of the alpha-chain COOH-terminal residue, Arg141 alpha) have been studied in the same crystal form by high resolution X-ray diffraction methods and by polarized absorption microspectrophotometry. Specifically, the crystal structure of deoxygenated desArg human hemoglobin has been refined at a 2.1 A resolution using crystals grown at low salt concentration from solutions of polyethylene glycol. The loss of Arg141 alpha and all of the salt bridges in which it participates is associated with subtle structural perturbations of the alpha-chains which include an increase in the conformational flexibility of both the NH2 and COOH-terminal peptides. Although the heme pockets appear unchanged and even the side-chain of Tyr140 is oriented nearly as in HbA, the functional characterization by microspectrophotometric measurements indicates that crystals of desArg hemoglobin bind oxygen with an affinity which is roughly 15-fold greater than that of crystals of human hemoglobin A. There is no alkaline Bohr effect or effect of chloride ions, but an acid Bohr effect is observed. The oxygen affinities measured along two principal axes of the crystals differ by 25%, indicating heterogeneity in the affinities of the oxygen binding sites. This finding and the measured Hill coefficient of unity suggest significant cooperativity in the binding of oxygen in these crystals. The origins of the observed heterogeneity and the implied cooperativity are unknown.

Allosteric effectors do not alter the oxygen affinity of hemoglobin crystals.

In solution, the oxygen affinity of hemoglobin in the T quaternary structure is decreased in the presence of allosteric effectors such as protons and organic phosphates. To explain these effects, as well as the absence of the Bohr effect and the lower oxygen affinity of T-state hemoglobin in the crystal compared to solution, Rivetti C et al. (1993a, Biochemistry 32:2888-2906) suggested that there are high- and low-affinity subunit conformations of T, associated with broken and unbroken intersubunit salt bridges. In this model, the crystal of T-state hemoglobin has the lowest possible oxygen affinity because the salt bridges remain intact upon oxygenation. Binding of allosteric effectors in the crystal should therefore not influence the oxygen affinity. To test this hypothesis, we used polarized absorption spectroscopy to measure oxygen binding curves of single crystals of hemoglobin in the T quaternary structure in the presence of the "strong" allosteric effectors, inositol hexaphosphate and bezafibrate. In solution, these effectors reduce the oxygen affinity of the T state by 10-30-fold. We find no change in affinity (< 10%) of the crystal. The crystal binding curve, moreover, is noncooperative, which is consistent with the essential feature of the two-state allosteric model of Monod J, Wyman J, and Changeux JP (1965, J Mol Biol 12:88-118) that cooperative binding requires a change in quaternary structure. Noncooperative binding by the crystal is not caused by cooperative interactions being masked by fortuitous compensation from a difference in the affinity of the alpha and beta subunits. This was shown by calculating the separate alpha and beta subunit binding curves from the two sets of polarized optical spectra using geometric factors from the X-ray structures of deoxygenated and fully oxygenated T-state molecules determined by Paoli M et al. (1996, J Mol Biol 256:775-792).

Preliminary crystal structure studies of a ternary electron transfer complex between a quinoprotein, a blue copper protein, and a c-type cytochrome.

A ternary electron transfer protein complex has been crystallized and a preliminary structure investigation has been carried out. The complex is composed of a quinoprotein, methylamine dehydrogenase (MADH), a blue copper protein, amicyanin, and a c-type cytochrome (c551i). All three proteins were isolated from Paracoccus denitrificans. The crystals of the complex are orthorhombic, space group C222(1) with cell dimensions a = 148.81 A, b = 68.85 A, and c = 187.18 A. Two types of isomorphous crystals were prepared: one using native amicyanin and the other copper-free apo-amicyanin. The diffraction data were collected at 2.75 A resolution from the former and at 2.4 A resolution from the latter. The location of the MADH portion was determined by molecular replacement. The copper site of the amicyanin molecule was located in an isomorphous difference Fourier while the iron site of the cytochrome was found in an anomalous difference Fourier. The MADH from P. denitrificans (PD-MADH) is an H2L2 hetero-tetramer with the H subunit containing 373 residues and the L subunit 131 residues, the latter containing a novel redox cofactor, tryptophan tryptophylquinone (TTQ). The amicyanin of P. denitrificans contains 105 residues and the cytochrome c551i contains 155 residues. The ternary complex consists of one MADH tetramer with two molecules of amicyanin and two of c551i, forming a hetero-octamer; the octamer is located on a crystallographic diad. The relative positions of the three redox centers--i.e., the TTQ of MADH, the copper of amicyanin, and the heme group of c55li--are presented.

Abnormal regulation of the hypothalamo-pituitary-adrenal axis in the genetically obese fa/fa rat.

Adrenalectomy has been shown to reverse most facets of the syndrome of the genetically obese fa/fa rat. However, a detailed analysis of the hypothalamo-pituitary-adrenal (HPA) axis in these animals is lacking. In the present study, morning corticosteronemia was higher in obese rats of both sexes than in lean ones, whereas evening corticosteronemia was higher only in obese male rats. The HPA axis was further investigated using stressful stimuli. Immobilization, ether, and cold stresses resulted in greater corticosterone levels in obese than in lean animals. These abnormalities consisted in upward shifts of the corticosterone response in obese females and absolute increases in that of obese males, indicating that such alterations were more pronounced in obese male than obese female rats. Due to this, the putative origin of the increased corticosterone output of obese rats was studied in males. Greater levels of ACTH were reached in obese than in lean rats when submitted to a cold stress (6 C). Dexamethasone produced a complete suppression of corticosterone output in both lean and obese rats. During the recovery from such suppression, corticosterone levels rose to higher values in obese than in lean rats. This observation together with the greater cold-induced ACTH output in obese rats suggest that the increased activity of the HPA axis of these animals is of central origin. Whatever its precise etiology within the central nervous system, it is proposed that the increased HPA axis activity in obese rats and its resultant hypercorticism play a role in the establishment and maintenance of their syndrome.

Changes in the hypothalamo-pituitary-adrenal axis of genetically obese fa/fa rats: a structural, immunocytochemical, and morphometrical study.

Most metabolic disorders of genetically obese Zucker rats are reversed by adrenalectomy and are restored by corticosterone treatment, thus suggesting that a functional hypercorticosteronemic state is involved in the pathogenesis of the obesity syndrome in fa/fa rats. However, the hormone content and morphology of the hypothalamo-pituitary-adrenal axis of this animal model have to our knowledge not yet been described. We, thus, investigated morphologically and morphometrically the hypothalamic regions involved in CRF synthesis and secretion in male fa/fa rats. To ascertain if the brain is selectively or uniformly affected, we studied the main nuclei of the lateral and mediobasal hypothalamus, i.e. arcuate, lateral hypothalamic, and ventromedial nucleus and the parvicellular portion of the paraventricular nucleus. Moreover, after immunocytochemical labeling, we analyzed densitometrically the CRF-bearing axons of the median eminence and the ACTH-containing cells of the anterior and intermediate lobe of the pituitary gland. Finally, we investigated the adrenal glands by qualitative light microscopy. In fa/fa rats most hypothalamic nuclei were structurally changed. Furthermore, hypothalamic CRF and anterior pituitary ACTH contents as well as adrenal weight were increased, the zona fasciculata of the adrenal cortex was hypertrophic, and the ACTH content of the intermediate lobe was reduced. In conclusion, our results demonstrate that the obesity syndrome in genetically obese fa/fa rats is associated with lesions of the hypothalamo-pituitary-adrenal axis consistent with hyperadrenocorticism due to hyperactivity of the whole adrenal axis. Alterations also occur in the hypothalamic nuclei controlling glycemia, insulinemia, and circadian corticosterone secretion.

One month of streptozotocin-diabetes induces different neuroendocrine and morphological alterations in the hypothalamo-pituitary axis of male and female rats.

LHRH (median eminence) and LH (pituitary and plasma) from male and female Sprague-Dawley rats were assayed 1 month after streptozotocin injection and compared with values in controls either fed ad libitum or offered a restricted diet. Plasma LH was also assayed after stimulation with exogenous LHRH or naloxone. In diabetic males, the median eminence LHRH content and the plasma LH response to exogenous LHRH were unaltered, pituitary LH was increased, and plasma LH was decreased under basal conditions and after naloxone treatment. In diabetic females, while the median eminence LHRH content and the plasma LH response to exogenous LHRH or naloxone were reduced, pituitary and plasma LH levels were not different. Measurements made in undernourished rats excluded the possibility that the alterations found in diabetic animals were nutrition dependent. In parallel experiments, hypothalami and pituitaries were examined morphologically. In diabetic animals, degenerate axons, mainly of the LHRH type, were found in the arcuate nucleus and median eminence, and LH gonadotrophs were altered and more numerous. Strong differences between control males and females were revealed by morphometry; moreover, diabetic females had higher brain weights and fewer LH gonadotroph changes than diabetic males. These studies indicate that 1) the hypothalamo-pituitary changes that occur early in our streptozotocin-treated rats are unrelated to undernourishment and are possibly caused by insulin deficiency; 2) the LHRH axonal lesions might play a primary pathogenic role in the hypothalamo-pituitary disorder; 3) some anatomical data indicate that the brain and pituitary are less severely affected by diabetes in female than in male animals; and 4) differences between control males and females may account for some of the dissimilarities between the sexes observed under diabetic conditions.

Stimulation of growth hormone release by thyrotropin-releasing hormone in the hypophysectomized rat bearing an ectopic pituitary.

Hypophysectomized female rats which received renal grafts of anterior pituitary (AP) or weight-matched intact controls were sampled under urethane anesthesia. Plasma growth hormone (GH) in sequential samples from each rat was measured by radioimmunoassay to determine the effect of exogenous thyrotropin-releasing hormone (TRH) on GH release from ectopic or intact AP. In a first experiment, following a baseline sample, a pre-treatment sample was taken from each rat 30 min after urethane injection, after which TRH (0.3 or 0.6 mug) or isotonic saline was injected iv, and samples were taken at 10 and 30 min post-treatment. Baseline GH levels in hypophysectomized-transplanted rats were in the range of 4.0 to 8.0 ng/ml, and were not modified significantly by urethane. TRH caused a significantly greater increase in growth hormone at 10 min than did saline. Plasma GH tended to be higher at 30 min post-treatment only in the 0.6 mug TRH-treated group. In further experiments the above described protocol was followed except that four doses of TRH were used (0.15, 0.3, 0.6, and 1.2 mug) and post-TRH blood samples were taken at 5 and 10 min. TRH caused a clear-cut increase in plasma GH both at 5 and 10 min, although no dose-effect relationship was present. In intact controls, baseline GH levels were in the range 40.0 to 80.0 ng/ml and were drastically reduced by urethane. In these animals, only the 1.2 mug TRH dose induced a GH rise at 5 and 10 min. In similar experiments, iv administration of vasopressin (100, 200, or 400 mU) induced a rise in plasma GH when given to the hypohysectomized-transplanted rats, but was ineffective in intact controls; the administration of prostaglandin E2 (5.0 and 50.0 mug) increased plasma GH in both experimental conditions. The results indicate that TRH in the hypophysectomized-transplanted rat acts directly on the AP tissue to increase GH release and that the ectopic pituitary is more susceptible than the in situ pituitary to some GH-releasing stimuli.

Growth hormone releasing activity of thyrotropin-releasing hormone in rats with hypothalamic lesions.

The growth hormone (GH)-releasing effect of thyrotropin-releasing hormone (TRH) was investigated in rats in which central nervous system (CNS)-anterior pituitary (AP) connections had been experimentally interrupted. Sprague-Dawley (SD) female and male rats, underwent bilateral electrolytic lesions in the median eminence (ME) or the ventromedial nuclei (VMN) or were sham-operated (sham-op). Fifteen days after surgery, 0.9% NAACl or TRH was injected iv into sham-op rats or those with lesions in the CNS, anesthetized with urethane, and blood was drawn at 5 and 10 min posttreatment. In the rats with ME lesions, TRH at all the doses used (0.1, 0.4 and 0.8 microng/100 g BW) induced a marked, although not dose-related GH rise, which was not present in sham-op rats after TRH, or after NaCl administration to either rats with ME lesions or sham-op rats. In SD male rats lesioned in the VMN, TRH at doses of 0.4 and 0.8 microng/100 g BW induced significant GH rises, while the lowest TRH dose (0.1 microng/100 g BW) was ineffective; again, TRH was ineffective at all doses used in sham-op rats. Concomitant evaluation of the prolactin (PRL)-releasing effect of TRH (0.1-0.8 microng/100 g BW), showed a striking elevation of plasma PRL in both female and male sham-op controls, but no PRL rise in the rats with ME lesions. The results reveal that in the rat with surgical separation of the anterior pituitary from the CNS, a direct GH-releasing effect of TRH can be obtained, whereas its PRL-releasing effect is no longer observed, and suggest that, by analogy, the GH-releasing effect of TRH present in some disease states of the human may be due to an impairment of CNS-AP connections.

Tuberoinfundibular dopaminergic neurons and lactotropes in young and old female rats.

Aging in female rats is accompanied by several endocrine dysfunctions, such as reproductive decline associated with characteristic hyperprolactinemia, lactotrope hyperplasia, and functional impairment of hypothalamic tuberoinfundibular dopaminergic (TIDA) neurons. The aim of this morphometrical, immunocytochemical, and densitometrical study was to gain a better anatomical knowledge of TIDA neurons and axons as well as of lactotropes in old female rats with (A) or without (NA) pituitary adenomas, compared with young animals. At the hypothalamic level, we found that tyrosine hydroxylase (TH)-labeled neurons in the arcuate nucleus were comparable in young and old NA yet their size and TH-content were increased in A animals. Also the TH-labeled median eminence axons did not differ significantly between young and old NA but were more numerous in the old A rats. Independently from adenomas, both number of prolactin (PRL)-labeled structures and content of immunoreactive PRL were increased in pituitaries of old rats, the plasma PRL levels, however, were high only in A. Our findings support the documented lactotrope hypertrophy and hyperplasia in old female rats and suggest that TIDA-neuron changes only occur in hyperprolactinemic animals carrier of adenomas.

Deacylation kinetics of gamma-chymotrypsin in solution and in the crystal.

The rate of catalyzed hydrolysis of the acyl enzyme analogs indolacryloyl-gamma-chymotrypsin and furylacryloyl-gamma-chymotrypsin in the crystal has been measured by single crystal microspectrophotometry and compared with the rate of catalyzed hydrolysis of acyl-gamma-chymotrypsin in solution and acyl-alpha-chymotrypsin both in solution and in the crystal. The maximal deacylation rate is the same for both species and independent of the physical state. However, the pH dependence of the deacylation rate of crystalline acyl-gamma-chymotrypsin shows a 0.9 unit shift in the pK of the catalytic system which is unique and probably consequent to specific lattice interactions.

Sexually dimorphic effects of aging on tuberoinfundibular dopaminergic neurons and lactotropes of rats.

The effect of aging on plasma prolactin (PRL) levels, hypothalamic tuberonifundibular dopaminergic (TIDA) neurons and pituitary lactotropes was evaluated in prolactinoma-free young (5-month-old) and old (23- to 24-month-old) Long-Evans rats of either sex. The young female rats were in diestrus, the old ones in recurrent pseudo-pregnancy. The tyrosine hydroxylase (TH)-labelled neurons in the arcuate nucleus (AN) and axons in the median eminence (ME) as well as the PRL-labelled lactotropes in the pituitary gland were studied by morphometry and densitometric immunohistochemistry. Further, we investigated the secretory function of isolated lactotropes by reverse hemolytic plaque assay (RHPA) and by cell culture in comparable animal groups. Compared with young animals, the plasma PRL levels of old rats of both sexes were similar or reduced. All morphometric and densitometric measurements of the AN neurons, ME axons (except number) and pituitary lactotropes were comparable in young and old female rats. In old male rats the AN and ME measurements were mostly decreased, while the lactotropes remained almost unchanged. The RHPA generally showed a reduced PRL release from lactotropes of old animals of both sexes. The PRL release from the cultured lactotropes, on the contrary, was greatly increased in old female rats and unchanged in old male rats. Our functional and morphological data suggest that the in vivo function of lactotropes in old prolactinoma-free female and male rats does not seem to be strongly influenced by the mildly reduced TIDA neuron activity, yet emphasize the differences of the aging process in the two sexes.

Carrier detection in Duchenne muscular dystropy.

We investigated the number of echinocytes, the serum hemopexin level, and spectrin band II phosphorylation in the blood of normal subjects, patients, and carriers of Duchenne dystrophy. The patients and carriers exhibited quantitatively significant differences with respect to controls.

Deformed erythrocytes in muscular dystrophies.

Because of previous reports of a possible correlation between echinocytogenesis and muscular dystrophies, we investigated the time-dependent development of echinocytes in the blood of normal subjects, patients, and healthy carries of Duchenne dystrophy. There was a quantitatively significant increase of echinocytes in patients and carriers.

The reduction of circulating growth hormone and prolactin in streptozocin-induced diabetic male rats is possibly caused by hypothalamic rather than pituitary changes.

To gain further information on diabetes-related disorders in the somatotrophic and lactotrophic axes, we undertook a functional, morphometrical and densitometrical study of the arcuate nucleus (AN), median eminence (ME) and anterior pituitary gland of adult male rats one month after streptozocin-induced diabetes (STZ-D). The basal secretory activity of somatotrophs and lactotrophs was tested by the reverse haemolytic plaque assay (RHPA) and plasma GH and prolactin (PRL) levels were determined by RIA. The number of GH-releasing factor (GRF)-labelled axons and the amount of axonal tyrosine hydroxylase (TH)-immunoreactivity increased in STZ-D. There were no significant differences in any of the other densitometrical measurements performed on GRF-, somatostatin-, thyrotropin-releasing hormone- and TH-labelled ME axon cross-sections as well as those on tuberoinfundibular-dopaminergic neurones of the AN in STZ-D compared with control rats. Plasma GH and PRL levels and measurements on anterior pituitary GH- and PRL-labelled structures were decreased in STZ-D. However, the GH and PRL plaque areas were increased after RHPA implying that the secretory capacity of somatotrophs and lactotrophs was not impaired. Taken together, these results suggest that the accumulated GRF in the ME is due to reduced GRF release. This could account for the reduced amplitude and/or frequency of GH secretory pulses. The increased axonal TH-immunoreactivity may indicate an increased dopamine synthesis. If coupled to increased release this could, in turn, be partly responsible for the reduced plasma and anterior pituitary PRL concentration.(ABSTRACT TRUNCATED AT 250 WORDS)

Thyrotrophin releasing hormone-induced growth hormone and prolactin release: physiological studies in intact rats and in hypophysectomized rats bearing an ectopic pituitary gland.

To determine how the sensitivity of the ectopic anterior pituitary gland to the GH-releasing effect of thyrotrophin releasing hormone (TRH) might be affected by the time lapse from transplantation, TRH (0-15 and 0-6 microng) was injected i.v. into hypophysectomized (hypox)-transplanted rats under urethane anaesthesia 1,3,8,15,30 and 60 days after transplantation, and plasma samples were taken 5 and 10 min later. Baseline GH values gradually decreased with time from about 16-0 ng/ml (1 day) to about 3-0 ng/ml (30 and 60 days). The TRH-induced GH release was absent 1 day after transplantation, present only with the higher TRH dose 3 and 8 days after transplantation, and clearly elicitable, also with the lower TRH dose (0-15 microng), from 15 up to 60 days. Determination of plasma prolactin concentrations showed a decline from about 85-0 ng/ml (1 day) to about 32-0 ng/ml (8 days); subsequently (15-60 days) prolactin values stabilized. Plasma prolactin levels increased 15 and 60 days after transplantation only when a dose of 0-6 microng TRH was given. In intact weight-matched rats, TRH induced a GH response only at the dose of 1-2 microng while a short-lived but clear-cut prolactin response could be obtained even with the 0-3 microng dose. The present results indicate that: (1) disconnexion between the central nervous system and the anterior pituitary gland greatly enhances GH responsiveness while blunting prolactin responsiveness to TRH; (2) the sensitivity of the anterior pituitary gland to the GH-releasing effect of TRH increases with time from transplantation; (3) TRH is a more effective prolactin- than GH-releaser on the pituitary gland in situ.