Growth hormone (GH)-releasing activity of chicken GH-releasing hormone (GHRH) in chickens.

Two peptides with sequence similarities to growth hormone releasing hormone (GHRH) have been identified by analysis of the chicken genome. One of these peptides, chicken (c) GHRH-LP (like peptide) was previously found to poorly bind to chicken pituitary membranes or to cloned and expressed chicken GHRH receptors and had little, if any, growth hormone (GH)-releasing activity in vivo or in vitro. In contrast, a second more recently discovered peptide, cGHRH, does bind to cloned and expressed cGHRH receptors and increases cAMP activity in transfected cells. The possibility that this peptide may have in vivo GH-releasing activity was therefore assessed. The intravenous (i.v.) administration of cGHRH to immature chickens, at doses of 3-100 µg/kg, significantly increased circulating GH concentrations within 10 min of injection and the plasma GH levels remained elevated for at least 30 min after the injection of maximally effective doses. The plasma GH responses to cGHRH were comparable with those induced by human (h) or porcine (p) GHRH preparations and to that induced by thyrotropin releasing hormone (TRH). In marked contrast, the i.v. injection of cGHRH-LP had no significant effect on circulating GH concentrations in immature chicks. GH release was also increased from slaughterhouse chicken pituitary glands perifused for 5 min with cGHRH at doses of 0.1 µg/ml or 1.0 µg/ml, comparable with GH responses to hGHRH1-44. In contrast, the perifusion of chicken pituitary glands with cGHRH-LP had no significant effect on GH release. In summary, these results demonstrate that cGHRH has GH-releasing activity in chickens and support the possibility that it is the endogenous ligand of the cGHRH receptor.

Modified alternate-day fasting regimens reduce cell proliferation rates to a similar extent as daily calorie restriction in mice.

Calorie restriction (CR) and alternate-day fasting (ADF) reduce cancer risk and reduce cell proliferation rates. Whether modified ADF regimens (i.e., allowing a portion of energy needs to be consumed on the fast day) work, as well as true ADF or CR to reduce global cell proliferation rates, remains unresolved. Here, we measured the effects of true ADF, modified ADF, and daily CR on cell proliferation rates in mice. Thirty female C57BL/6J mice were randomized to one of five interventions for 4 wk: 1) CR-25% (25% reduction in daily energy intake), 2) ADF-75% (75% reduction on fast day), 3) ADF-85% (85% reduction on fast day), 4) ADF-100% (100% reduction on fast day), and 5) control (ad libitum intake). Body weights of the ADF groups did not differ from controls, whereas the CR-25% group weighed less than all other groups posttreatment. Epidermal cell proliferation decreased (P<0.01) by 29, 20, and 31% in the CR-25%, ADF-85% and ADF-100% groups, respectively, relative to controls. Proliferation rates of splenic T cells were reduced (P<0.01) by 37, 32, and 31% in the CR-25%, ADF-85%, and ADF-100% groups, respectively, and mammary epithelial cell proliferation was 70, 65, and 62% lower (P<0.01), compared with controls. Insulin-like growth factor-1 levels were reduced (P<0.05) in the CR-25% and ADF-100% groups only. In summary, modified ADF, allowing the consumption of 15% of energy needs on the restricted intake day, decreases global cell proliferation similarly as true ADF and daily CR without reducing body weight.

Chronic changes in peripheral growth hormone levels do not affect ghrelin stomach mRNA expression and serum ghrelin levels in three transgenic mouse models.

Ghrelin is an endogenous ligand for the growth hormone secretagogue (GHS) receptor. Ghrelin is involved in feeding behaviour and is a potent stimulator of GH release. Chronically increased GH concentrations are known to negatively regulate the pituitary GHS receptor. This study tested whether chronic changes in peripheral GH levels/action affect ghrelin mRNA expression and circulating concentrations of ghrelin. Stomach ghrelin mRNA expression and serum concentrations of ghrelin were measured in three groups of transgenic mice and the respective control animals: group 1, GH-receptor gene disrupted mice (GHR/KO); group 2, mice expressing bovine GH (bGH); and group 3, mice expressing GH-antagonist (GHA). Ghrelin mRNA expression in the stomach, pituitary and hypothalamus of young adult male rats were measured using reverse-transcription-polymerase chain reaction. Ghrelin mRNA expression levels were approximately 3000-fold higher in rat stomach than in rat pituitary. Ghrelin mRNA expression in rat hypothalamus was below the detection limits of our assay. Stomach ghrelin mRNA expression, as well as serum concentrations of ghrelin, did not change significantly in any of the three mouse groups compared to the respective control group. These data support previous observations that the stomach is the main source of circulating ghrelin, and also indicate that stomach ghrelin mRNA expression and serum concentrations of ghrelin are not affected by chronic changes in peripheral GH/insulin-like growth factor-I levels/action.

Molecular cloning of ovine and bovine growth hormone-releasing hormone receptors: the ovine receptor is C-terminally truncated.

To provide information about species differences in GH-releasing hormone (GHRH) receptors useful for studies of receptor-ligand binding properties and receptor function, we have cloned the ovine and bovine pituitary GHRH receptors (GHRHRs). The ovine receptor (oGHRHR) was cloned from a pituitary complementary DNA library and encodes a protein that is similar to that of porcine, human, rat, and mouse with, respectively, 84.3, 80.7, 75.9, and 74.0% amino acid identity. Surprisingly, oGHRHR has a 16 amino acid truncation at its carboxyl-terminal end when compared with GHRHRs from other known mammals. RT-PCR using pooled pituitary RNA from a different population of sheep could detect only truncated receptor. Bovine GHRHR (bGHRHR) was cloned by RT-PCR and shows 92.5% amino acid sequence identity with oGHRHR, but has no truncation. Genomic sequencing of the appropriate region of goat receptor intron 13 showed that the caprine receptor shares the same truncation seen in sheep. Photoaffinity cross-linking of GHRH to ovine and bovine pituitary membranes confirms that the native ovine pituitary GHRHR protein is smaller by the amount predicted by the cloned sequences. The truncation did not affect GHRH binding as oGHRHR, bGHRHR, human GHRHR, and human GHRHR, which was truncated by site-directed mutagenesis to match the oGHRHR, all showed comparable GHRH binding affinity when expressed in transfected cell lines. In contrast, the ovine and truncated human receptors demonstrated enhanced sensitivity for GHRH stimulation of cAMP (lowered ED(50)) relative to hGHRHR and bGHRHR. This suggests that this C-terminal domain acts to inhibit cAMP signaling possibly through a role in receptor down regulation.

Intracerebroventricular administration of the rat growth hormone (GH) receptor antagonist G118R stimulates GH secretion: evidence for the existence of short loop negative feedback of GH.

Pulsatile growth hormone (GH) secretion is regulated by three hypothalamic factors, growth hormone-releasing hormone (GHRH), somatostatin and the natural ligand for the GH secretagogue receptor (Ghrelin). These factors and their effects are, in turn, affected by short loop feedback of GH itself. To test the hypothesis that hypothalamic GH receptors are involved in the ultradian rhythmicity of pituitary GH secretion, the rat GH receptor antagonist (G118R) was administered to adult male rats by intracerebroventricular (i.c. v.) injection and the effects on spontaneous GH secretion were studied. Normal saline was administered i.c.v. to eight control rats. Mean GH concentrations increased significantly in the rat treated with G118R compared to rats that received normal saline. The pulse amplitude rose by a mean of 33.3 ng/ml and the total area under the curve increased by a mean of 15 061 ng/ml x min. The number of GH peaks did not change significantly following G118R. These data suggest that GH regulates its own secretion by acting directly on hypothalamic GH receptors.

High plasma growth hormone (GH) levels inhibit expression of GH secretagogue receptor messenger ribonucleic acid levels in the rat pituitary.

Synthetic GH secretagogues (GHSs) act via a receptor (GHS-R) distinct from that of GH-releasing hormone. The GHS-R has been cloned from the pituitary and is expressed not only in the pituitary but also in specific areas of the brain, including the hypothalamus. Recent studies suggest that hypothalamic GHS-R expression is regulated by GH. This study was designed to investigate whether pituitary GHS-R expression is modulated by GH. Female Wistar-Furth rats were injected sc with either saline (control) or GC tumor cells (GC) that secrete rat GH. The tumors were allowed to develop for 1-4 weeks. At weeks 1-4, control (n = 4-8) and GC rats (n = 3-8) were killed. Pituitary GHS-R messenger RNA (mRNA) was measured by a quantitative competitive PCR assay. The endogenous GHS-R mRNA levels were measured by determining the amount of competitive template RNA required to produce equimolar amounts of native and competitive template PCR products. The mean log plasma GH levels were significantly greater in the GC rat group than in the control group at weeks 2, 3, and 4. At these times, the mean log pituitary GHS-R mRNA contents were significantly lower in the GC rat group than in the control group. No relationship could be established between log estradiol levels and GHS-R levels. These data indicate that pituitary GHS-R expression is modulated by GH.

Growth hormone-releasing hormone stimulates mitogen-activated protein kinase.

GH-releasing hormone (GHRH) can induce proliferation of somatotroph cells. The pathway involving adenylyl cyclase/cAMP/protein kinase A pathway in its target cells seems to be important for this action, or at least it is deregulated in some somatotroph pituitary adenomas. We studied in this work whether GHRH can also stimulate mitogen-activated protein (MAP) kinase. GHRH can activate MAP kinase both in pituitary cells and in a cell line overexpressing the GHRH receptor. Although both protein kinase A and protein kinase C could activate MAP kinase in the CHO cell line studied, neither protein kinase A nor protein kinase C appears to be required for GHRH activation of MAP kinase in this system. However, sequestration of the betagamma-subunits of the G protein coupled to the receptor inhibits MAP kinase activation mediated by GHRH. This pathway also involves p21ras and a phosphatidylinositol 3-kinase, probably phosphatidylinositol 3-kinase-gamma. Despite the involvement of p21ras, the protein kinase Raf-1 is not hyperphosphorylated in response to GHRH, contrary to what usually occurs when the Ras-Raf-MAP kinase pathway is activated. In summary, this work describes for the first time the activation of MAP kinase by GHRH and outlines a path for this activation that is different from the cAMP-dependent mechanism that has been traditionally described as mediating the mitogenic actions of GHRH.

The mutant growth hormone-releasing hormone (GHRH) receptor of the little mouse does not bind GHRH.

The little mouse is a dwarf strain characterized by low levels of GH, pituitary hypoplasia, and an unresponsiveness to treatment with exogenous GHRH. The defect has been mapped to a missense mutation in the GHRH receptor gene that abolishes the function of the receptor, but the mechanism of this inactivation is unknown. Receptor function might be affected at the level of protein expression, maturation and processing, localization to the cell surface, ligand binding, or signaling. In this study, Western blots, using antiserum raised against the GHRH receptor and immunoprecipitation analysis of epitope-tagged receptors, demonstrate that both wild-type and mutant receptor proteins are expressed at equivalent levels in transfected cells. Immunofluorescence analysis of intact and permeabilized cells expressing the epitope-tagged receptors suggests that wild-type and little mouse receptors are similarly localized to the cell surface. A species homologous binding assay was developed and used to show that 125I-mouse GHRH binds with high affinity to the wild-type mouse receptor but not to the little mutant receptor. Consistent with this, the mutant receptor fails to stimulate intracellular cAMP accumulation. Our results demonstrate that the little mutation does not dramatically affect the expression level, glycosylation, or cellular localization of the receptor protein but that it blocks specific GHRH binding, and therefore, signaling does not take place.

Molecular and cell biology of the growth hormone-releasing hormone receptor.

The GHRH receptor is a seven transmembrane G-protein-linked receptor found predominantly in the pituitary gland. It is essential for normal somatotroph proliferation and for the synthesis and secretion of GH. Significant amounts of GHRH receptor are also found in the hypothalamus, kidney and placenta. Transcription of the GHRH receptor gene promoter is enhanced by Pit-1 and by glucocorticoids but is inhibited by oestrogen. Recently, mutations involving severe GHRH receptor truncations have been associated with human type 1 GHD. Studies of chimeric receptors and of GHRH receptor cross-linking sites have shown that the N-terminal extracellular domain of the GHRH receptor is required for hormone binding, but that key sites for ligand specificity and signalling are associated with the transmembrane helices and intervening loops. Evidence from the ovine GHRH receptor suggests that the C-terminus has an inhibitory function and may be involved in down-regulation via internalization and phosphorylation. A better understanding of the GHRH receptor may lead to new therapies for the treatment of GH disorders.

Overexpression of the growth-hormone-releasing hormone gene in acromegaly-associated pituitary tumors. An event associated with neoplastic progression and aggressive behavior.

The clinical behavior of growth hormone (GH)-producing pituitary tumors is known to vary greatly; however, the events underlying this variability remain poorly understood. Herein we demonstrate that tumor overexpression of the GH-releasing hormone (GHRH) gene is one prognostically informative event associated with the clinical aggressiveness of somatotroph pituitary tumors. Accumulation of GHRH mRNA transcripts was demonstrated in 91 of a consecutive series of 100 somatotroph tumors by in situ hybridization; these findings were corroborated by Northern analysis and reverse transcriptase polymerase chain reaction, and protein translation was confirmed by Western blotting. By comparison, transcript accumulation was absent or negligibly low in 30 normal pituitary glands. GHRH transcripts were found to preferentially accumulate among clinically aggressive tumors. Specifically, GHRH mRNA signal intensity was 1) linearly correlated with Ki-67 tumor growth fractions (r = 0.71; P < 0.001), 2) linearly correlated with preoperative serum GH levels (r = 0.56; p = 0.01), 3) higher among invasive tumors (P < 0.001), and 4) highest in those tumors in which post-operative remission was not achieved (P < 0.001). Using multivariate logistic regression, a model of postoperative remission likelihood was derived wherein remission was defined by the single criterion of suppressibility of GH levels to less than 2 ng/ml during an oral glucose tolerance test. In this outcome model, GHRH mRNA signal intensity proved to be the most important explanatory variable overall, eclipsing any and all conventional clinicopathological predictors as the single most significant predictor of postoperative remission; increases in GHRH mRNA signal were associated with marked declines in remission likelihood. The generalizability of this outcome model was further validated by the model's significant performance in predicting postoperative remission in a random sample of 30 somatotroph tumors treated at another institution. These data indicate that overexpression of GHRH gene is an event associated with the neoplastic progression and clinical aggressiveness of somatotroph adenomas. More generally, these data merge essential elements of the hypothalamic and pituitary hypotheses of pituitary tumorigenesis, providing for a more unified concept of neoplastic progression in the pituitary.

Growth hormone-releasing hormone receptor mRNA in acromegalic pituitary tumors.

The growth hormone (GH)-releasing hormone receptor (GHRH-R) has been recently cloned and found to be a member of a new family of seven transmembrane receptors that includes secretin, vasoactive intestinal peptide, calcitonin, and corticotropin-releasing factor. GHRH-R mRNA has been demonstrated by Northern blot analyses to be present specifically in the anterior pituitary gland. To determine the precise cellular localization of this receptor in normal anterior pituitary and pituitary adenomas, GHRH-R mRNA was analyzed in 2 normal human pituitary glands and 16 human pituitary adenomas using in situ hybridization. GHRH-R was specifically localized in somatotroph cells in the normal pituitary. In the adenomas, all GH-producing adenomas originating from acromegalic patients demonstrated up-regulation of GHRH-R mRNA when compared with levels in the normal pituitary. Only one of five clinically nonfunctioning adenomas, a gonadotroph luteinizing hormone/follicle-stimulating hormone-positive adenoma, exhibited up-regulation of this receptor message. Adrenocorticotrophic hormone-secreting and prolactin-secreting adenomas did not express GHRH-R message. In summary, GHRH-R is specifically expressed in somatotrophs and GH-producing adenomas, suggesting that GHRH-R may influence GH release in adenomas similar to this receptor's actions in the normal somatotrophs and may be involved in the growth of GH-secreting adenomas.

Growth hormone-releasing hormone and growth hormone-releasing peptide as therapeutic agents to enhance growth hormone secretion in disease and aging.

Growth hormone (GH) secretion is pulsatile and is tightly regulated. In this chapter the effects of aging, nutrition, the feedback effects of IGF-I, and the role of body composition in the decline of GH secretion will be discussed. In GH-deficient adults there is an increase in the amount of intra-abdominal (visceral) fat. Similarly, with increasing age, there is an increase in visceral fat and there is a tight correlation between 24-hour GH release and visceral fat in the elderly. This may have serious metabolic consequences, including insulin resistance and increased cardiovascular risk. There are at least four potential mechanisms for the age-related decline in GH secretion: 1) decreased release of growth hormone releasing-hormone (GHRH); 2) increased release of somatostatin; 3) enhanced sensitivity to IGF-I feedback; and 4) decreased somatotroph mass. The latter two potential mechanisms are discussed. There is little evidence that there is any change in sensitivity to IGF-I feedback with aging and the somatotroph cell mass appears to be preserved in older subjects. The GH axis may be stimulated by either GHRH or by growth hormone-releasing peptide (GHRP) and related compounds. Chronic therapy with GHRH in GH-deficient children restores GH secretion and accelerates linear growth. Mutations of the GHRH receptor lead to GH deficiency and short stature. This indicates the essential role of GHRH in regulation of GH secretion. Growth hormone releasing peptide was discovered in 1981. Recently, the GHRP/GH secretagogue receptor has been cloned and orally active GHRP mimetics have been developed. One such compound, MK-677, stimulates pulsatile GH secretion and its effects persist for 24 hours. Oral administration of MK-677 for a month in the elderly demonstrates that this route stimulates a physiologic pattern of GH secretion. The amplitude of the GH pulses was increased but the number of GH pulses was unchanged. Thus, in older individuals, the amount of GH secreted in 24 hours is restored toward that seen in young adults. This compound also enhances GH secretion in GH-deficient adults who had been GH-deficient during childhood. The development of stable, orally active molecules to stimulate the GHRP/GH secretagogue receptor is a practical reality. These GH secretagogues may have a therapeutic role in short stature and adult GH deficiency. In addition, the use of GH secretagogues in normal aging merits investigation, as growth hormone may regulate body composition in older adults.

Distribution of alpha 2C-adrenergic receptor-like immunoreactivity in the rat central nervous system.

The distribution of alpha 2C-adrenergic receptors (ARs) in rat brain and spinal cord was examined immunohistochemically by using an affinity purified polyclonal antibody. The antibody was directed against a recombinant fusion protein consisting of a 70-amino-acid polypeptide portion of the third intracellular loop of the alpha 2C-AR fused to glutathione-S-transferase. Selectivity and subtype specificity of the antibody were demonstrated by immunoprecipitation of [125I]-photoaffinity-labeled alpha 2-AR and by immunohistochemical labeling of COS cells expressing the individual rat alpha 2-AR subtypes. In both cases the antibody recognized only the alpha 2C-AR subtype, and immunoreactivity was eliminated by preadsorption of the antibody with excess antigen. In rat brain, alpha 2C-AR-like immunoreactivity (alpha 2C-AR-LI) was found primarily in neuronal perikarya, with some labeling of proximal dendrites; analysis by confocal microscopy revealed the intracellular localization of some of the immunoreactivity. Areas of dense immunoreactivity include anterior olfactory nucleus, piriform cortex, septum, diagonal band, pallidum, preoptic areas, supraoptic nucleus, suprachiasmatic nucleus, paraventricular nucleus, amygdala, hippocampus (CA1 and dentate gyrus), substantia nigra, ventral tegmental area, raphe (pontine and medullary), motor trigeminal nucleus, facial nucleus, vestibular nucleus, dorsal motor nucleus of the vagus, and hypoglossal nucleus. Labeling was found in specific laminae throughout the cortex, and a sparse distribution of very darkly labeled cells was observed in the striatum. At all levels of the spinal cord there were small numbers of large, darkly labeled cells in layer IX and much smaller cells in layer X. In general, the pattern of alpha 2C-LI throughout the neuraxis is consistent with previously published reports of the distribution of receptor mRNA detected by hybridization histochemistry.

Purification of the growth hormone releasing hormone receptor with a C-terminal, biotinylated affinity ligand.

The receptor for growth hormone-releasing hormone (GHRH) has been purified from bovine pituitary tissue and HEK293 cells transfected with human or porcine receptor using a retrievable biotinylated GHRH analog. Custom synthesized [His1, Nle27, Biotin-Lys41]-human GHRH-(1-41)-NH2 (GHRHb) bound to pituitary membranes with affinity comparable to human GHRH. GHRHb which has the biotinyl group on the C-terminus of the peptide allowed simultaneous binding to both the receptor and streptavidin agarose. This analog was used directly in the purification of the receptor from pituitary tissue or was modified by incorporation of the photoaffinity group ANBNOS (GHRHlambdab), radioiodinated and used to demonstrate purification of the GHRH receptor from transfected HEK293 cell membranes. Membranes were prepared and prebound with the respective ligand followed by CHAPS-solubilization and application of the solubilized complex to a streptavidin agarose column. Analysis of eluates from the pituitary tissue purification by silver stained SDS PAGE or of autoradiographs of gels from HEK293 eluates revealed specific bands of 52 and 55 kDa, respectively. The higher size of the latter band is expected for the ligand-crosslinked receptor. Both bands displayed similar mobility shifts of 10 kDa upon treatment with N-glycosidase, a method previously used to characterize this receptor. A 45 kDa band corresponding to the size of the Gs alpha subunit was also detected in eluates of the silver stained gels, suggesting that the GHRH receptor was retrieved as a heterotrimeric complex. Fold purification and yield for this procedure were estimated to be greater than 50,000 and 2.6-9%, respectively.

Photoaffinity cross-linking to the pituitary receptor for growth hormone-releasing factor.

Photoaffinity cross-linking methods presented here demonstrate a 55-kilodalton (kDa) GH-releasing factor (GRF) receptor in ovine pituitary membranes and in cell lines expressing the cloned human pituitary receptor complementary DNA. Covalent cross-linking of photoprobe to this high affinity site is strongly competed by 1 nM GRF. Competition shows strong specificity for GRF over related peptides. Reduced cross-linking in the presence of guanosine 5'-O-(3-thiotriphosphate) suggests that this is a G-protein-coupled receptor. Detection of cross-linking to this receptor required detergent extraction to reduce high nonspecific binding of GRF photoprobe. Partial deglycosylation of the cross-linked receptor with neuraminidase caused a shift in apparent size to 52 kDa. Complete deglycosylation with N-glycosidase caused a shift to 45 kDa, demonstrating that this receptor is an N-linked glycoprotein and agreeing with the protein size and single glycosylation site predicted from the cloned complementary DNA sequence. These sizes differ from those found in previous reports which used chemical cross-linking to identify GRF receptor. This photoaffinity cross-linking method will facilitate studies of receptor function and tissue distribution. Photoaffinity cross-linking can also be used to map regions of the receptor molecule and bound GRF that are in close proximity.

Molecular cloning and expression of a human anterior pituitary receptor for growth hormone-releasing hormone.

GH-releasing hormone (GHRH), acting through the GHRH receptor (GHRH-R), plays a pivotal role in the regulation of GH synthesis and secretion in the pituitary. It is possible that GHRH may serve other roles in other tissues. Here we report the cloning of a cDNA encoding a human GHRH-R from an acromegalic pituitary cDNA library. The isolated cDNA encodes a 423-amino acid protein that has seven putative transmembrane domains characteristic of G-protein-coupled receptors. It is a member of the secretin family of G-protein-coupled receptors and has 47%, 42%, 35%, and 28% identity with receptors for vasoactive intestinal peptide, secretin, calcitonin, and PTH, respectively. Transient expression of this cDNA in COS cells induced saturable, high affinity, GHRH-specific binding and also stimulated intracellular cAMP accumulation in response to physiological concentrations of GHRH. A specific GHRH antagonist blocked both binding and second messenger response. Northern analysis indicated that GHRH-R mRNA was most abundant in extracts of pituitary and was not detected in other tissues.

Seasonal changes in the activation of crossbridge motions of isolated thick filament from Limulus striated muscle.

In dynamic light scattering, measurements of the intensity-intensity time correlation function from a suspension of rod-like particles of length L could reveal dynamical information related to translational and internal motions of those particles. For a suspension of thick filaments isolated from the myosin-regulated, striated muscles of Limulus at KL greater than 1 (where K is the scattering vector), the average characteristic linewidth (gamma) increased with the addition of Ca2+ or with the depletion of ATP. The increase in the gamma with the addition of Ca2+ could be due to the presence of energy-requiring, high-frequency motions of the crossbridges activated by Ca2+. The increase in gamma which occurred with the depletion of ATP was assumed to be mainly due to the thermal motions of the crossbridges after they had moved radially away from the filament backbone. The percentage increase in gamma following the addition of Ca2+ was found to be seasonal, i.e., values of gamma obtained from thick filaments isolated between the middle of June and the middle of September were smaller than those obtained during the rest of the year. The effect of temperature on the percentage increase in gamma was also different. The increase showed a maximum at about 35 degrees C during the summer and at about 25 degrees C at other times. However, the percentage increase in gamma developed under ATP-depleted conditions showed no temperature-related maximum. The number of bound Ca2+ per myosin molecule was 1 during the summer and 2 at other times.

G-protein-mediated Ca2+ sensitization of smooth muscle contraction through myosin light chain phosphorylation.

The Ca2+ sensitivities of tonic (pulmonary and femoral artery) and phasic (portal vein and ileum) smooth muscles and the effects of guanosine 5'-O-(gamma-thiotriphosphate) (GTP gamma S) and norepinephrine on Ca2+ sensitivity of force development and myosin light chain (MLC20) phosphorylation were determined in permeabilized preparations that retained coupled receptors and endogenous calmodulin. The Ca2+ sensitivity of force was higher (approximately 3-fold) in the tonic than in the phasic smooth muscles. The nucleotide specificity of Ca2+ sensitization was: GTP gamma S much greater than GTP greater than ITP much greater than CTP = UTP. Baseline phosphorylation (7% at pCa greater than 8) and maximal phosphorylation (58% at pCa 5.0) were both lower in portal vein than in femoral artery (20 and 97%). Norepinephrine and GTP gamma S increased phosphorylation at constant [Ca2+] (pCa 7.0-6.5). MLC20 phosphorylation induced by norepinephrine was completely inhibited by guanosine 5'-O-(beta-thiodiphosphate) (GDP beta S). In portal vein at pCa 5, GTP gamma S increased phosphorylation from 58%, the maximal Ca2(+)-activated value, to 75%, and at pCa greater than 8, from 7 to 13%. In femoral artery at pCa 5, neither phosphorylation (97%) nor force was affected by GTP gamma S, while at pCa greater than 8, GTP gamma S caused an increase in force (16% of maximum) with a borderline increase in MLC20 phosphorylation (from 20 to 27%). MLC20 phosphorylation (up to 100%) was positively correlated with force. The major results support the hypothesis that the G-protein coupled Ca2(+)-sensitizing effect of agonists on force development is secondary to increased MLC20 phosphorylation.