Deterministic construct of amplifying actions of ghrelin on pulsatile growth hormone secretion

Ghrelin is a native ligand for the growth hormone secretagogue (GHS) receptor that stimulates pulsatile GH secretion markedly. At present, no formal construct exists to unify ensemble effects of ghrelin, GH-releasing hormone (GHRH), somatostatin (SRIF), and GH feedback. To model such interactions, we have assumed that ghrelin can stimulate pituitary GH secretion directly, antagonize inhibition of pituitary GH release by SRIF, oppose suppression of GHRH neurons in the arcuate nucleus (ArC) by SRIF, and induce GHRH secretion from ArC. The dynamics of such connectivity yield self-renewable GH pulse patterns mirroring those in the adult male and female rat and explicate the following key experimental observations. 1) Constant GHS infusion stimulates pulsatile GH secretion. 2) GHS and GHRH display synergy in vivo. 3) A systemic pulse of GHS stimulates GH secretion in the female rat at any time and in the male more during a spontaneous peak than during a trough. 4) Transgenetic silencing of the neuronal GHS receptor blunts GH pulses in the female. 5) Intracerebroventricular administration of GHS induces GH secretion. The minimal construct of GHS-GHRH-SRIF-GH interactions should aid in integrating physiological data, testing regulatory hypotheses, and forecasting innovative experiments.     

A construct of interactive feedback control of the GH axis in the male

Growth hormone (GH) secretion is controlled by GH-releasing hormone (GHRH), the GH release-inhibiting hormone somatostatin (SRIF), and autofeedback connections. The ensemble network produces sexually dimorphic patterns of GH secretion. In an effort to formalize this system, we implemented a deterministically based autonomous feedback-driven construct of five principal dose-responsive regulatory interactions: GHRH drive of GH pituitary release, competitive inhibition of GH release by SRIF, GH autofeedback via SRIF with a time delay, delayed GH autonegative feedback on GHRH, and SRIF inhibition of GHRH secretion. This formulation engenders a malelike pattern of successive GH volleys due jointly to positive time-delayed feedback of GH on SRIF and negative feedback of SRIF on GH and GHRH. The multipeak volley is explicated as arising from a reciprocal interaction between GH and GHRH during periods of low SRIF secretion. The applicability of this formalism to neuroendocrine control is explored by initial parameter sensitivity analysis and is illustrated for selected feedback-dependent experimental paradigms. The present construct is not overparameterized and does not require an ad hoc pulse generator to achieve pulsatile GH output. Further evolution of interactive constructs could aid in exploring more complex feedback postulates that confer the vivid sexual dimorphism of female GH profiles.     

Joint pituitary-hypothalamic and intrahypothalamic autofeedback construct of pulsatile growth hormone secretion

Growth hormone (GH) secretion is vividly pulsatile in all mammalian species studied. In a simplified model, self-renewable GH pulsatility can be reproduced by assuming individual, reversible, time-delayed, and threshold-sensitive hypothalamic outflow of GH-releasing hormone (GHRH) and GH release-inhibiting hormone (somatostatin; SRIF). However, this basic concept fails to explicate an array of new experimental observations. Accordingly, here we formulate and implement a novel fourfold ensemble construct, wherein 1) systemic GH pulses stimulate long-latency, concentration-dependent secretion of periventricular-nuclear SRIF, thereby initially quenching and then releasing multiphasic GH volleys (recurrent every 3-3.5 h); 2) SRIF delivered to the anterior pituitary gland competitively antagonizes exocytotic release, but not synthesis, of GH during intervolley intervals; 3) arcuate-nucleus GHRH pulses drive the synthesis and accumulation of GH in saturable somatotrope stores; and 4) a purely intrahypothalamic mechanism sustains high-frequency GH pulses (intervals of 30-60 min) within a volley, assuming short-latency reciprocal coupling between GHRH and SRIF neurons (stimulatory direction) and SRIF and GHRH neurons (inhibitory direction). This two-oscillator formulation explicates (but does not prove) 1) the GHRH-sensitizing action of prior SRIF exposure; 2) a three-site (intrahypothalamic, hypothalamo-pituitary, and somatotrope GH store dependent) mechanism driving rebound-like GH secretion after SRIF withdrawal in the male; 3) an obligatory role for pituitary GH stores in representing rebound GH release in the female; 4) greater irregularity of SRIF than GH release profiles; and 5) a basis for the paradoxical GH-inhibiting action of centrally delivered GHRH.     

Unequal autonegative feedback by GH models the sexual dimorphism in GH secretory dynamics

Growth hormone (GH) secretion, controlled principally by a GH-releasing hormone (GHRH) and GH release-inhibiting hormone [somatostatin (SRIF)] displays vivid sexual dimorphism in many species. We hypothesized that relatively small differences within a dynamic core GH network driven by regulatory interactions among GH, GHRH, and SRIF explain the gender contrast. To investigate this notion, we implemented a minimal biomathematical model based on two coupled oscillators: time-delayed reciprocal interactions between GH and GHRH, which endow high-frequency (40-60 min) GH oscillations, and time-lagged bidirectional GH-SRIF interactions, which mediate low-frequency (occurring every 3.3 h) GH volleys. We show that this basic formulation, sufficient to explain GH dynamics in the male rat [Farhy LS, Straume M, Johnson ML, Kovatchev BP, and Veldhuis JD. Am J Physiol Regulatory Integrative Comp Physiol 281: R38-R51, 2001], emulates the female pattern of GH release, if autofeedback of GH on SRIF is relaxed. Relief of GH-stimulated SRIF release damps the slower volleylike oscillator, allowing emergence of the underlying high-frequency oscillations that are sustained by the GH-GHRH interactions. Concurrently, increasing variability of basal somatostatin outflow introduces quantifiable, sex-specific disorderliness of the release process typical of female GH dynamics. Accordingly, modulation of GH autofeedback on SRIF within the interactive GH-GHRH-SRIF ensemble and heightened basal SRIF variability are sufficient to transform the well-ordered, 3.3-h-interval, multiphasic, volleylike male GH pattern into a femalelike profile with irregular pulses of higher frequency.     

Estrogen and androgen elicit growth hormone release via dissimilar patterns of hypothalamic neuropeptide secretion

The dimorphic pattern of growth hormone (GH) secretion and somatic growth in male and female mammals is attributable to the gonadal steroids. Whether these hormones mediate their effects solely on hypothalamic neurons, on somatotropes or on both to evoke the gender-specific GH secretory patterns has not been fully elucidated. The purpose of this study was to determine the effects of 17beta-estradiol, testosterone and its metabolites on release of GH, GH-releasing hormone (GHRH) and somatostatin (SRIF) from bovine anterior pituitary cells and hypothalamic slices in an in vitro perifusion system. Physiological concentrations of testosterone and estradiol perifused directly to anterior pituitary cells did not affect GH releases; whereas, dihydrotestosterone and 5alpha-androstane-3alpha, 17beta-diol increased GH. Perifusion of testosterone at a pulsatile rate, and its metabolites and estradiol at a constant rate to hypothalamic slices in series with anterior pituitary cells increased GH release. The androgenic hormones increased GHRH and SRIF release from hypothalamus; whereas, estradiol increased GHRH but decreased SRIF release. Our data show that estradiol and the androgens generated distinctly different patterns of GHRH and SRIF release, which in turn established gender-specific GH patterns.     

Model-projected mechanistic bases for sex differences in growth hormone regulation in humans

Models of physiological systems facilitate rational experimental design, inference, and prediction. A recent construct of regulated growth hormone (GH) secretion interlinks the actions of GH-releasing hormone (GHRH), somatostatin (SRIF), and GH secretagogues (GHS) with GH feedback in the rat (Farhy LS, Veldhuis JD. Am J Physiol Regul Integr Comp Physiol 288: R1649-R1663, 2005). In contrast, no comparable formalism exists to explicate GH dynamics in any other species. The present analyses explore whether a unifying model structure can represent species- and sex-defined distinctions in the human and rodent. The consensus principle that GHRH and GHS synergize in vivo but not in vitro was explicable by assuming that GHS 1) evokes GHRH release from the brain, 2) opposes inhibition by SRIF both in the hypothalamus and on the pituitary gland, and 3) stimulates pituitary GH release directly and additively with GHRH. The gender-selective principle that GH pulses are larger and more irregular in women than men was conferrable by way of 4) higher GHRH potency and 5) greater GHS efficacy. The overall construct predicts GHRH/GHS synergy in the human only in the presence of SRIF when the brain-pituitary nexus is intact, larger and more irregular GH pulses in women, and observed gender differences in feedback by GH and the single and paired actions of GHRH, GHS, and SRIF. The proposed model platform should enhance the framing and interpretation of novel clinical hypotheses and create a basis for interspecies generalization of GH-axis regulation.     

Enhanced growth hormone responses to growth hormone releasing hormone in male type I diabetic patients.

In a previous paper we have demonstrated that growth hormone (GH) responses to growth hormone releasing hormone (GHRH) are higher in premenopausal normal women than in age matched healthy men. As in type I diabetes mellitus various disturbances of GH secretion have been reported, the aim of our study was to assess the effect of sex on basal and GHRH stimulated GH secretion in type I diabetes mellitus. In 21 female and 23 male type I diabetic patients and 28 female and 30 male control subjects GH levels were measured before and after stimulation with GHRH (1 microgram/kg body weight i.v.) by radioimmunoassay. GH responses to GHRH were significantly higher in female than in male control subjects (p less than 0.02), whereas the GH levels following GHRH stimulation were similar in female and male type I diabetic patients. GH responses to GHRH were significantly higher in the male type I diabetic patients than in the male control subjects (p less than 0.001); in the female type I diabetic patients and the female control subjects, however, GH responses to GHRH were not statistically different. The absence of an effect of sex on GHRH stimulated GH responses in type I diabetes mellitus provides further evidence of an abnormal GH secretion in this disorder.     

Central somatostatinergic regulation of growth hormone secretion in dwarf chickens.

1. Basal circulating growth hormone (GH) concentrations in sex-linked-dwarf (SLD) chickens were unaffected by the intracerebroventricular (icv) injection of 10, 50 or 100 micrograms somatostatin (SRIF). 2. The GH response to systemic thyrotropin-releasing hormone (TRH; 10 micrograms/kg, iv) was, however, 'paradoxically' enhanced 20 min after icv SRIF administration. 3. A lower dose (1.0 micrograms) of SRIF had no effect on basal or TRH-induced GH release. 4. High-titre SRIF antisera (4 microliters) also had no acute effect on basal plasma GH concentrations, but augmented the GH response to TRH challenge. 5. SRIF would appear to act at central sites to modulate stimulated GH secretion in SLD chickens.     

Robust growth hormone (GH) secretion in aged female rats co-administered GH-releasing hexapeptide (GHRP-6) and GH-releasing hormone (GHRH)

Aging is associated with a blunted growth hormone (GH) secretory response to GH-releasing hormone (GHRH), in vivo. The objective of the present study was to assess the effects of aging on the GH secretory response to GH-releasing hexapeptide (GHRP-6), a synthetic GH secretagogue. GHRP-6 (30 micrograms/kg) was administered alone or in combination with GHRH (2 micrograms/kg) to anesthetized female Fischer 344 rats, 3 or 19 months of age. The peptides were co-administered to determine the effect of aging upon the potentiating effect of GHRP-6 on GHRH activity. The increase in plasma GH as a function of time following administration of GHRP-6 was lower (p less than 0.001) in old rats than in young rats; whereas the increase in plasma GH secretion as a function of time following co-administration of GHRP-6 and GHRH was higher (p less than 0.001) in old rats than in young rats (mean Cmax = 8539 +/- 790.6 micrograms/l vs. 2970 +/- 866 micrograms/l, respectively; p less than 0.01). Since pituitary GH concentrations in old rats were lower than in young rats (257.0 +/- 59.8 micrograms/mg wet wt. vs. 639.7 +/- 149.2 micrograms/mg wet wt., respectively; p less than 0.03), the results suggested that GH functional reserve in old female rats was not linked to pituitary GH concentration. The differential responses of old rats to individually administered and co-administered GHRP-6 are important because they demonstrate that robust and immediate GH secretion can occur in old rats that are appropriately stimulated. The data further suggest that the cellular processes subserving GH secretion are intact in old rats, and that age-related decrements in GH secretion result from inadequate stimulation, rather than to maladaptive changes in the mechanism of GH release.     

Induction of growth hormone (GH) mRNA by pulsatile GH-releasing hormone in rats is pattern specific

Growth hormone-releasing hormone (GHRH) is a main inducer of growth hormone (GH) pulses in most species studied to date. There is no information regarding the pattern of GHRH secretion as a regulator of GH gene expression. We investigated the roles of the parameters of exogenous GHRH administration (frequency, amplitude, and total amount) upon induction of pituitary GH mRNA, GH content, and somatic growth in the female rat. Continuous GHRH infusions were ineffective in altering GH mRNA levels, GH stores, or weight gain. Changing GHRH pulse amplitude between 4, 8, and 16 microg/kg at a constant frequency (Q3.0 h) was only moderately effective in augmenting GH mRNA levels, whereas the 8 microg/kg and 16 microg/kg dosages stimulated weight gain by as much as 60%. When given at a 1.5-h frequency, GHRH doubled the amount of GH mRNA, elevated pituitary GH stores, and stimulated body weight gain. In the rat model, pulsatile but not continuous GHRH administration is effective in inducing pituitary GH mRNA and GH content as well as somatic growth. These studies suggest that the greater growth rate, pituitary mRNA levels, and GH stores seen in male compared with female rats are likely mediated, in part, by the endogenous episodic GHRH secretory pattern present in males.     

Reduced Somatostatin in Hypothalamus of Young Male Mouse Increases Local but not Circulatory GH

The release of growth hormone (GH) from the pituitary gland is primarily inhibited by somatostatin (SRIF) from the hypothalamus via interactions with five types of SRIF receptors (SSTRs). However, the inhibition mechanism of SRIF on GH has not been fully examined. In this study, we repressed the hypothalamic SRIF in young male mice by stereotaxic injection of the lentiviral-shRNA against SRIF to investigate the role of hypothalamic SRIF on hormone secretion in the GH/IGF-1 axis. We found that the reduction of SRIF in hypothalamus was associated with an increase in the protein, but not the mRNA level, of the GH in the pituitary where SSTR 2 and SSTR 5 act importantly. Interestingly, the level of blood circulatory SRIF, GH, IGF-1 and the body weight were not significantly influenced by the downregulation of hypothalamic SRIF. Our findings provide insights into the mechanisms underlying the inhibition of SRIF on GH secretion.     

Single exposure to testosterone in adulthood rapidly induces regularity in the growth hormone release process

The neonatal gonadal steroid milieu is known to be important in imprinting the striking sexual dimorphism of growth hormone (GH) secretion; however, the influence of the sex steroids on GH control in adult life and their mechanism/site of action are largely unknown. In the present study, we tested the hypothesis that testosterone (T) subserves the gender-specific regularity of the GH release process in adulthood. The approximate entropy statistic (ApEn) was used to quantify the degree of regularity of GH release patterns over time. Eighteen hours after a single subcutaneous injection of 1 mg T, both sham-operated and ovariectomized (OVX) female adult rats displayed plasma GH profiles that were strikingly similar to the regular male-like ultradian rhythm of GH secretion. The highest ApEn values, denoting greater disorderliness of GH secretion, were observed in the ovary-intact group, and T injection significantly (P < 0.001) reduced this irregularity whether or not the ovaries were present. Serial intravenous injections of GH-releasing hormone (GHRH) caused a similar increase in plasma GH levels in sham-operated females independently of time of administration. In contrast, female rats administered T exhibited a male-like intermittent pattern of GH responsiveness to GHRH, the latter known to be due to the cyclic release of endogenous somatostatin. These results demonstrate that acute exposure to T during adult life can rapidly and profoundly "masculinize" GH pulse-generating circuits in the female rat. Our findings suggest that the enhanced orderliness characteristic of the GH release process in males, compared with females, is regulated by T. We postulate that this T-induced regularity is mediated at the level of the hypothalamus by inducing regularity in somatostatin secretion, which in turn governs overall GH periodicity.     

Estradiol modulation of growth hormone secretion in the ewe: no growth hormone-releasing hormone neurons and few somatotropes express estradiol receptor alpha

Evidence suggests that estrogen modulates growth hormone (GH) release and that GH plays an important role in follicular and ovulatory processes. How estradiol affects GH secretion is unclear. Having verified that there is a coincident surge of GH at the time of the preovulatory LH surge, immunocytochemical studies incorporating high-temperature antigen retrieval were used to determine whether GH-releasing hormone (GHRH) neurons, somatotropes, or both, expressed estrogen receptor alpha (ER), in the ewe. Although GHRH neurons were surrounded by many ER cells, they did not express immunocytochemically detectable ERs. In contrast to gonadotropes, in which the majority expressed ERs, few somatotropes were estrogen receptive. These data suggest that estrogen does not act directly on GHRH neurons to influence GH secretion, and any direct effect on pituitary GH release, through the ERalpha, may be small.     

Insulin-induced hypoglycemia, L-dopa and arginine stimulate GH secretion through different mechanisms in man

We sought to clarify the mechanisms of growth hormone (GH) secretion induced by insulin hypoglycemia, L-dopa, and arginine in man. The secretion of GH as measured by increased plasma level, in response to oral administration of 500 mg L-dopa or 30 min-infusion of arginine, was not modified by prior intravenous administration of 200 micrograms GH-releasing hormone (GHRH). It was, however, completely blocked by preadministered 50 micrograms SMS201-995, a long-acting somatostatin (SRIH) analog. GH release with 200 micrograms GHRH was completely blocked by 100 micrograms SMS201-995. GH secretion caused by insulin-induced hypoglycemia was significantly reduced but still present after administration of 100 micrograms of the analog. These results suggest that a suppression of SRIH release may be partially involved in the stimulatory mechanism of GH secretion by L-dopa. Coadministration of GHRH accentuated the stimulatory effect of arginine on GH secretion. Arginine significantly raised plasma TSH levels. These findings suggest that arginine suppresses SRIH release from the hypothalamus to cause GH secretion because SRIH suppresses TSH secretion. It is also suggested that some factor (or factors) other than GHRH and SRIH are involved in the mechanism by which insulin-induced hypoglycemia stimulates GH secretion, because the effect of insulin was not fully blocked in the presence of SRIH analog. Thus all the tests for GH release appear to act via different mechanisms.     

Anatomy of the Hypophysiotropic Somatostatinergic and Growth Hormone-releasing Hormone System Minireview

The central control of growth hormone (GH) secretion from the pituitary gland is ultimately achieved by the interaction between two hypothalamic neurohormones, somatostatin which inhibits and growth hormone-releasing hormone (GHRH) which stimulates GH release. The regulation of the somatostatin and GHRH release from the hypothalamus is regulated by a range of other neuropeptides, neurotransmitters, neurohormones. In this mini review we attempt to provide a short summary covering the anatomy and chemical characteristics of the various cell populations regulating GH secretion as a tribute to Miklós Palkovits who pioneered the field of functional neuroanatomy of hypothalamic networks.Special Issue Dedicated to Miklós Palkovits.     

Masculinization of growth hormone (GH) secretory pattern by dihydrotestosterone is associated with augmentation of hypothalamic somatostatin and GH-releasing hormone mRNA levels in ovariectomized adult rats.

The role of androgen in the sexual dimorphism in hypothalamic growth hormone (GH)-releasing hormone (GHRH) and somatostatin (SS) gene expression was examined in rats. In the first study, the SS and GHRH mRNA levels were measured in both male and female rats at 4, 6, 8, and 10 weeks of age. A significant sex-related difference in the SS and GHRH mRNA levels was observed after 8 weeks of age, when sexual maturation is fully attained. Male rats had higher SS and GHRH mRNA levels than the female rats. In the second study, adult ovariectomized rats received daily injection of dihydrotestosterone (DHT), nonaromatizable testosterone, at a dose of 2 mg/rat for 21 days. The DHT treatment masculinized the GH secretory pattern, which was indistinguishable from that of intact male rats, and simultaneously augmented the SS and GHRH mRNA levels. The DHT treatment of ovariectomized rats after hypophysectomy significantly raised the level of SS mRNA, but not that of GHRH mRNA compared to the control animals. These findings suggest that the activation of the SS gene expression through androgen receptor plays an important role in the maintenance of sexual dimorphism in GH secretion in rats.     

Regulation of galanin secretion from pituitary cells in vitro by estradiol and GHRH

The effects of estradiol and growth hormone-releasing hormone (GHRH) on galanin release from anterior pituitary cells were examined in vitro. 17-β-Estradiol (0.001–10 nM) increased galanin secretion from anterior pituitary cells in a concentration-dependent manner. Estradiol (10 nM) increased galanin release 300 and 600% from pituitary cells of ovariectomized and male rats, respectively. Immunocytochemical studies demonstrated that estradiol (10 nM) increased the number of galanin-containing cells twofold after 4 days in culture. Growth hormone-releasing hormone (1 and 10 nM) increased and SRIF (1 and 10 nM) decreased galanin release from pituitary cells of ovariectomized and male rats. We conclude that estradiol increases galanin release by a direct effect on pituitary cells, in part by increasing the number of pituitary cells synthesizing galanin. In addition, GHRH stimulates galanin release when estradiol levels are low.     

alpha-Glycerylphosphorylcholine administration increases the GH responses to GHRH of young and elderly subjects.

Growth hormone (GH) secretion is decreased during aging in humans and in rodents. This decrease may be due to increased hypothalamic somatostatin release, which is inhibited by cholinergic agonists, or to decreased secretion of GHRH. Alpha-glyceryl-phosphorylcholine (alpha-GFC) is a putative acetylcholine precursor used in the treatment of cognitive disorders in the elderly. In order to learn what effect alpha-GFC had on GH secretion, GH-release hormone (GHRH) was given to young and old human volunteers, with or without the addition of alpha-GFC. GH secretion was greater in the younger subjects than in the old individuals, and both groups had a greater GH response to the GHRH+alpha-GFC than to GHRH alone. The potentiating effect of alpha-GFC on GH secretion was more pronounced in the elderly subjects. These findings confirm the observation that aged individuals respond less well to GHRH than younger subjects, and provides further evidence that increased cholinergic tone enhances GH release.     

Age-related differences in growth hormone (GH) regulation during strenuous exercise.

This study was designed to investigate the central neuroendocrine mechanisms by which exercise (EX) stimulates growth hormone (GH) release as a function of age. Twelve male subjects, six in their early-to-mid twenties and six in their late sixties or seventies, received a strong GH stimulus either as incremental EX until volitional exhaustion or by administration of GHRH alone or Hex alone two hours after a presumed maximal GH response to combined administration of GHRH plus hexarelin (Hex). Total GH availability was calculated as area under the curve (AUC) over time periods 0 - 120 and 120 - 240 min. The mean AUC in micro g/l x 120 min to GHRH+Hex in the younger group was approximately twice that in the older group (11,260, range 3,947 - 19,007 vs. 5,366, range 2,262 - 8,654). In younger males, the mean AUC to EX (509, range 0 - 1,151) was larger than to GHRH (119, range 0 - 543), but less than that to Hex (919, range 0 - 1,892). In the older group, GH responses to EX and GHRH were abolished (mean AUC: 112, range 0 - 285, and 156, range 30 - 493), respectively) in contrast to the response to Hex (1,077, range 189 - 1,780). These data indicate that maximal GH stimulation by GHRH+Hex results in greater desensitization of GHRH compared to Hex, irrespective of age. We postulate that the abolished responsiveness of GH to EX in older group is due to insufficient disinhibition of hypothalamic somatostatin activity and desensitization of GHRH, while the preserved activity of a central Hex-related pathway is not involved. The GH response to EX in younger males is due to complete inhibition of somatostatin activity and stimulation of a central Hex-related pathway in spite of GHRH desensitization. We conclude that a central Hex-related pathway is the primary factor for EX-induced GH release only in younger males.     

Growth hormone secretion: molecular and cellular mechanisms and in vivo approaches

Growth hormone (GH) release is under the direct control of hypothalamic releasing hormones, some being also produced peripherally. The role of these hypothalamic factors has been understood by in vitro studies together with such in vivo approaches as stalk sectioning. Secretion of GH is stimulated by GH-releasing hormone (GHRH) and ghrelin (acting via the GH secretagogue [GHS] receptor [GHSR]), and inhibited by somatostatin (SRIF). Other peptides/proteins influence GH secretion, at least in some species. The cellular mechanism by which the releasing hormones affect GH secretion from the somatotrope requires specific signal transduction systems (cAMP and/or calcium influx and/or mobilization of intracellular calcium) and/ or tyrosine kinase(s) and/or nitric oxide (NO)/cGMP. At the subcellular level, GH release (at least in response to GHS) is accomplished by the following. The GH-containing secretory granules are moved close to the cell surface. There is then transient fusion of the secretory granules with the fusion pores in the multiple secretory pits in the somatotrope cell surface.