Minimally-invasive anterior maxillary distraction technique in patients with cleft lip and palate and maxillary deficiency: an evaluation of 106 patients.

Our aim was to evaluate the feasibility of a minimally-invasive surgical technique for anterior maxillary distraction osteogenesis to correct maxillary hypoplasia in patients with clefts. A modified Y distractor was placed intraorally in 106 patients with cleft- associated maxillary deficiency to facilitate protraction of the maxilla. Subsequently the patients had an anterior maxillary osteotomy through a minimally invasive incision, followed by activation of the appliance at the rate of 0.8mm/day until positive overjet was achieved. The patient's lateral cephalograms were evaluated preoperatively (T1), after activation (T2), and one year postoperatively (T3). Collected data were assessed with the paired t test, and probabilities of < 0.001 were accepted as significant. A mean (SD) of 10.4 (2.58) mm anterior maxillary advancement was obtained in all patients after 10-13 days of distraction. The sella-nasion-point A (SNA) angle increased from 75.37° to 83.01°. When we compared the cephalometric variables at T1 and T2, the mean maxillary length and overjet at T2 were significantly higher (p<0.001). The comparison of mean values at T2 and T3 was not significant. Minimally invasive anterior maxillary distraction with the modified Y distractor resulted in changes after activation that were consistent one year postoperatively, making it a conservative, less traumatic, and effective treatment of cleft-related maxillary deficiency.

Clinico-pathomorphological, serum biochemical and histological studies in broilers fed ochratoxin A and a toxin deactivator (Mycofix Plus).

1. Toxic effects of two concentrations (0.5 and 1 mg/kg) of ochratoxin A (OTA) and attenuating effects of a toxin deactivator (Mycofix Plus(MTV INSIDE)) containing the yeast Trichosporon mycotoxinivorans on the performance (feed conversion ratio; body weight gain), serum enzymes (lactate dehydrogenase, gamma-glutamyltranspeptidase and aspartate aminotransferase) and clinico-pathomorphology of internal organs were studied in 270 one-day-old broiler chicks divided into 9 groups over a 42-d period. 2. Feed conversion ratios (FCR) in groups fed toxin deactivator were improved compared with groups receiving OTA only. An increase in the relative weight of kidney and liver was observed in groups fed 0.5 and 1 mg/kg OTA on day 42 of the experiment as compared with the control group. In contrast, relative weights of bursa of Fabricius and spleen were not significantly affected in experimental groups exposed to OTA as compared to control groups determined on days 28 and 42 of age. 3. Serum enzymes (LDH, GGT and AST) values in OTA treated groups determined on days 28 and 42 were higher than those of the control group. 4. Histopathological examination of kidney on day 42 revealed degenerative changes in the epithelial cells of the proximal convoluted tubules and massive necrosis of the proximal tubular epithelial cells. These changes were less marked in birds receiving 0.5 mg/kg OTA than in those receiving 1 mg/kg. In general, histological changes in kidneys, liver, bursa and spleen were less pronounced in birds receiving OTA and toxin deactivator concomitantly. 5. Dietary OTA at 0.5 and 1 mg/kg adversely affects FCR, increases the serum liver enzymes and induces pronounced pathomorphological and histological changes in internal organs of broiler chicks. Co-administration of OTA with deactivator attenuated the harmful effects.

Clinical assessment of class II resin-based composites versus preformed metal crowns performed on primary molars in patients at high risk of caries.

AIM: To compare class II resin composite with preformed metal crowns (PMC) in the treatment of proximal dentinal caries in high caries-risk patients. METHODS: The charts (270) of paediatric patients with proximal caries of their primary molars were reviewed. Success or failure of a procedure was assessed using the dental notes. Survival analysis was used to calculate the mean survival time (MST) for both procedures. The influence of variables on the mean survival time was investigated. RESULTS: A total of 593 class II resin composites and 243 PMCs were placed in patients ranging between 4-13 years of age. The failure percentage of class II resin composites was 22.6% with the majority having been due to recurrent caries, while the failure percentage of PMCs was 15.2% with the majority due to loss of the crown. There was no significant difference between the MST of class II resin composites and PMCs, 41.3 and 45.6 months respectively (p value = 0.06). In class II resin composites, mesial restorations were associated with lower MST compared to distal restorations (p-value < 0.001). CONCLUSIONS: The MST of resin composites and PMCs were comparable when performed on high caries-risk patients.

Essential role of adenosine triphosphate in activation of 17beta-hydroxysteroid dehydrogenase in the rat Leydig cell.

The forskolin-induced steroidogenic block of testosterone production residing beyond pregnenolone synthesis in rat Leydig cells was localized to the level of the 17beta-hydroxysteroid dehydrogenase (17betaHSD) reaction in this study. The use of forskolin analogs that discriminate between the diterpene's inhibitory effect on the glucose transporter(s) (1,9-dideoxyforskolin) and its activation of adenylate cyclase (6-aminoethyl carbamyl forskolin) revealed that the block is related to inhibition of glucose transporter(s). 1,9-Dideoxyforskolin, but not 6-aminoethyl carbamyl forskolin, caused a significant inhibition of basal and hCG-stimulated testosterone production with accumulation of androstenedione. Glucose-deficient media produced the same metabolic block in the absence of forskolin, with a significant reduction in 17betaHSD activity and increases in the apparent Km for androstenedione. In contrast, metabolic steps before testosterone formation were not affected. Glucose-induced 17betaHSD activation was mimicked by the addition of ATP or GTP in glucose-deficient media, but not by nonhydrolyzable triphosphate analogs or NADPH. A decrease in 17betaHSD activity caused by KT-5720, a specific inhibitor of protein kinase A and the calmodulin antagonist W-7, indicates that the ATP requirement may be related to the participation of protein kinases in the activation of 17betaHSD. ATP levels derived from alternative (nonglycolytic) pathways are adequate to support basal and hormone-stimulated enzymatic activities in the metabolism of cholesterol to androstenedione. However, the integrity of the glucose transport system with subsequent ATP generation is required for activation of 17betaHSD in the final step of androgen biosynthesis. In conclusion, the conversion of androstenedione to testosterone requires the contribution of the glycolytic pathway to meet ATP requirements for 17betaHSD activity.

The rat 17beta-hydroxysteroid dehydrogenase type III: molecular cloning and gonadotropin regulation.

17Beta-hydroxysteroid dehydrogenase (17betaHSD), the enzyme that catalyzes the final step of testosterone biosynthesis in the testis, was cloned from a rat Leydig cell complementary DNA library to gain insights into the functional requirements, activation mechanisms, and molecular regulation. The 17betaHSD complementary DNA encoded 306 amino acids (molecular mass of 33.7 kDa) and displayed 75% and 85% amino acid sequence homology to the human and mouse 17betaHSD type III enzymes, respectively. Northern analysis revealed a single 1.4-kb messenger RNA (mRNA) species in rat Leydig cells, whereas ovarian mRNA was detected only by RT-PCR amplification. The cloned 17betaHSD expressed in mammalian cell lines specifically catalyzed the reductive reaction in androgen formation with androstenedione as the preferred substrate. This reaction was significantly reduced in the absence of glucose. Expression of the endogenous 17betaHSD gene in rat Leydig cells was inhibited by a single dose of hCG in vivo, with maximum reduction of steady state mRNA levels at 24 h and recovery at 9 days. Such agonist-induced down-regulation of 17betaHSD expression, which preceded the marked reduction of LH receptors, resulted from changes at the transcriptional level and was accompanied by loss of enzymatic activity. These studies have demonstrated a glucose requirement for optimal activity of the enzyme in vitro and for a role of gonadotropin in regulating the expression of 17betaHSD gene in vivo. Cloning of the 17betaHSD type III enzyme from rat Leydig cells will facilitate further investigation of the molecular regulation of its activity in the testis.

A cAMP independent inhibitory action of high doses of forskolin in rat Leydig cells.

In addition to well known direct stimulatory and potentiatory actions of forskolin, we have previously reported that low doses of this diterpene (10(-9), 10(-12) M) markedly inhibit the production of cAMP and testosterone in rat Leydig cells through a pertussis toxin sensitive G-protein (A. Khanum and M. L. Dufau, J. Biol. Chem. 261, 1986). A different type of inhibitory effect of forskolin is described in this study. Forskolin (10(-5) M) markedly stimulates basal adenylate cyclase activity (about 200%) in rat Leydig cell membranes and potentiates the stimulatory effect of gonadotropin (10(-9), 10(-7) M) on adenylate cyclase in presence or in absence of GTP (10(-5) M). Similarly a time-dependent stimulation of forskolin (10(-5) M) alone is noted on all cAMP pools and testosterone production. Using a supramaximal steroidogenic dose of hCG (0.26 nM) or choleragen (0.1 microM), forskolin potentiates the gonadotrophin and toxin-induced responses of all cAMP pools significantly while inhibiting testosterone production. Moreover, forskolin also inhibits 8-Bromo-cAMP stimulated steroidogenesis. In contrast, pregnenolone synthesis was not altered by the diterpene. We have demonstrated in this study that the inhibitory effect of high doses of forskolin on steroidogenesis is distal to cAMP generation, and resulted from a steroidogenic block residing beyond pregnenolone synthesis.

Regulation of steroidogenic enzymes and a novel testicular RNA helicase.

Luteinizing hormone (LH) supports steroidogenesis and maintains testicular and ovarian function. Mediators of LH action exert homologous regulation of membrane receptors, steroidogenic enzymes and other regulatable genes of the Leydig cell (LC). Androgen and estrogen induced by LH could act through its cognate receptors in the LC to regulate gene expression. Although androgens are unquestionable essential for spermatogenesis and presumably exert their heterologous action through androgen receptors present in the Sertoli its regulatory mechanism in germinal cell maturation is far from clear. In contrast to physiological concentrations of gonadotropins which maintain the steroidogenic functions and LH and prolactin receptors in the gonads, high concentrations of gonadotropin (hCG) cause receptor down-regulation and desensitization of steroidogenic enzymes of the LCs in vivo (3beta-hydroxysteroid dehydrogenase types I and II, 17alpha-hydroxylase/17,20 lyase, and 17beta-hydroxysteroid dehydrogenase type III [17beta-HSD]). In addition, 17beta-HSD is regulated by compartmentalized endogenous glucose/ATP. The attenuation of steroidogenesis which results from receptor mediated activation by cognate hormone, but is independent of the subsequent phase of receptor down-regulation, is due to changes at the transcriptional level. Among the candidates affecting this regulation are active steroid metabolites (direct or indirect of steroids and other mediator(s) i.e. cAMP, putative transcription factors induced by LH action). Differential display assay revealed another gene which is transcriptionally regulated by gonadotropin termed GRTH (Gonadotropin Regulated Testicular Helicase). GRTH is a novel member of the DEAD-box family of RNA helicases, and is specifically expressed in LCs and meiotic LC of the testis. It is markedly up-regulated by hCG via cAMP-induced androgen formation in LCs at doses that cause down-regulation of receptors and steroidogenic enzymes. GRTH functions as a translational activator. Androgen produced by gonadotropin stimulation exerts intracrine/autocrine actions on GRTH, and also could influence transcription within the seminiferous tubule. GRTH may contribute to the control of steroidogenesis, including the restoration of down regulated cellular functions, and in the paracrine regulation of androgen dependent gene(s) involved in the meiotic process, and could thus have a crucial role in spermatogenesis.

Regulation of androgen synthesis: the late steroidogenic pathway.

Studies of the regulation of androgen synthesis in steroidogenic cells have focused on both transcriptional and post-translational regulation of the proteins that catalyze these reactions: the P450c17 that catalyzes the production of DHEA or androstenedione in consecutive hydroxylase and lyase activities, and the 17 beta-hydroxysteroid dehydrogenase (17 beta-HSD) that catalyzes the conversion of androstenedione to testosterone. Our studies of the regulation of the CYP17 lyase activity at the molecular level have utilized species- and tissue-specific differences to identify target regulatory sequences. Adenovirus infection of rat CYP17 promoter/luciferase reporter gene constructs in primary cultures of rat adrenal and rat Leydig cells revealed a rat-specific domain between-1 and -108 bp that cause inhibition of both basal and cAMP-induced CYP17 transcription in the adrenal, but not the Leydig cell. In contrast, similar promoter constructs from other species exhibited substantial cAMP-induced transcriptional activity in the rat adrenal. Mutagenesis of the conserved region of the rat and human proteins reveals significant differences in the amino acid domains required for hydroxylase and lyase activities within and between the two species, consistent with their differential regulation of lyase activity. The 17 beta-hydroxysteroid dehydrogenase (17 beta-HSD) reaction requires a viable glucose transporter system for optimal activity, and a high-energy phosphate was discovered to be the requisite product of glucose metabolism in 17 beta-HSD activation. These studies have provided insight into potential mechanisms of control of androgen synthesis in the late steroidogenic pathway, at the transcriptional and post-translational levels.

Breast carcinoma in Pakistani women.

In many developed and developing countries including Pakistan, breast cancer is the most frequently diagnosed cancer and the leading cause of cancer death in women. Among 4575 women presenting to the Cancer Research Foundation of Pakistan between 1987 and 1994 with breast lumps, 1201 (26%) were found to have breast cancer. Their ages ranged from 19 to 79 years. The peak incidence was in the 30 to 39 age group. Most patients were multiparous with an average of five children. The size of the tumor was greater than 5 cm in 66% of the cases. Invasive ductal carcinoma was the most common morphological type. According to the Bloom and Richardson grading system, 58% of cases were grade III. Lymph node metastases were present in 73% of the cases.

Effect of testosterone on epididymal proteins in castrated rhesus monkeys.

The effect of testosterone on regulation of epididymal protein synthesis has been investigated in castrated rhesus monkeys (Macaca mulatta). The proteins in the treated monkeys were characterized using polyacrylamide gel electrophoresis (under nondenaturing and denaturing conditions) and electrofocusing. At least four distinct proteins have been shown to be synthesized by the monkey epididymis under testosterone influence. Two of these proteins were detected following two days of testosterone treatment while the other two proteins were detected after a six-day treatment period. None of these proteins was detectable in monkeys treated with estradiol for six days. Electrofocusing of epididymal cytosol proteins from untreated and testosterone-treated and castrated monkeys also confirmed the presence of four androgen-dependent proteins in this species. The isoelectric points of these proteins were shown to range between 5.8 and 6.4. The molecular weights of these proteins were found to vary between 47,500 and 66,000. The in vitro incorporation of 3H-labeled amino acids was markedly greater in the androgen-primed epididymis as compared with the control tissue.

Health care utilization during terminal child illness in squatter settlements of Karachi.

OBJECTIVE: Information on health seeking behavior and health care utilization has important policy implications in health systems development. The paper presents some of the issues related to health care utilization and health seeking behavior in case of terminal child illness in seven squatter settlements of Karachi. METHODS: From seven squatter settlements of Karachi, with a population of 100,000 approximately, we collected information, using pretested structured questionnaire, from the mothers on health care utilization during the final illness of under five children dying during 1995-1996. These deaths were identified from an earlier baseline health and demographic survey in these areas. RESULTS: Interviews were completed for 259 infant and child deaths of which 57% were boys. Of all deaths 72% were taken to a health care provider, of which 82% went as soon as the child got ill. Private sector is the most preferred first choice i.e., 83%. Of all those who had been to a health care provider, 65% were referred to some other place and 72% of them took more than 12 hours altogether to reach the referred facility. Children in older age categories (OR 4.4 95% CI 2.22-8.67 and OR 5.0, 95% CI 2.09-12.31), boys (OR 2.6, 95% CI 1.46-4.77) and those with appropriate or incomplete immunization (OR 4.1, 95% CI 2.13-7.94) were significantly associated with the health care utilization as compared to their counterparts. CONCLUSION: Living in urban areas does not ensure accessibility to effective health care. In poor urban communities, referral to other facility delay the initiation of effective treatment in case of child illness leading to death which could be prevented otherwise. Private sector constitutes an important segment of our health care system, which requires strengthening and back up support. Furthermore, the study finding is suggestive of gender discrimination in health seeking behavior.

Effects of prostaglandin F2 alpha and its synthesis inhibitor indomethacin on corporaluteal functions in pseudopregnant rats.

The effects of PGF2 alpha and its synthesis inhibitor indomethacin on corporaluteal (CL) functions were studied in adult pseudopregnant rats. The CL functions were assessed by studying the duration of pseudopregnancy and histological changes in the ovary. Administration of PGF2 alpha (4 mg/kg BW) significantly (P less than 0.001) shortened the duration of pseudopregnancy. Histological examination of ovaries revealed regressed CL. Administration of indomethacin, on the other hand, significantly (P less than 0.001) prolonged the duration of pseudopregnancy. Histological examination of ovaries revealed large and well formed CL, the diameters of which were significantly (P less than 0.05) increased. Administration of indomethacin and PGF2 alpha simultaneously, however, keeps the duration of pseudopregnancy within normal limit. Further, the shortening in the duration of pseudopregnancy by PGF2 alpha alone was completely reversed by exogenous administration of progesterone. Since PGF2 alpha shortens and indomethacin (an inhibitor of PGF2 alpha synthesis) prolongs the duration of pseudopregnancy, it is concluded that PGF2 alpha acts as a luteolytic agent in rats. The mechanism of luteolysis is most likely to be due to decrease plasma progesterone level. An estimation of blood progesterone level after administration of PGF2 alpha to pseudopregnant rats is therefore, suggested.

Effect of chemotherapy on circulating steroid hormone levels in postoperative premenopausal breast cancer patients.

Serum levels of 17-beta oestradiol, testosterone and progesterone were determined in postoperative premenopausal breast cancer patients. In patients receiving chemotherapy circulating 17-beta oestradiol values decreased significantly compared to control group during the sampling/drug regimens employed. Among the control group, however, the oestradiol levels remained high throughout the sampling period. Testosterone levels in patients were also significantly low compared to control group throughout the sampling regimen up to 28 days. In contrast the levels of progesterone in patients were elevated and remained high compared to the corresponding controls. A positive correlation was found between the drop in serum oestradiol and testosterone levels following the initiation of chemotherapy and the regression of the tumour size. Steroid hormone levels in the serum of breast cancer patients receiving chemotherapy can serve as clinical tools to monitor the progress of the disease and response to therapy.

Angiotensin II receptors and inhibitory actions in Leydig cells.

Rat Leydig cells possess functional high-affinity receptors for angiotensin II (AII). AII inhibits adenylate cyclase activity in Leydig cell membranes and reduces basal and human chorionic gonadotropin (hCG)-stimulated cAMP pools and testosterone production in intact cells. Treatment of cells with an inhibitory dose of forskolin (10(-9) M) and a submaximal dose of AII caused additive inhibition of hCG-stimulated events. The inhibitory action of AII was largely prevented by pertussis toxin prior to the addition of AII alone or in the presence of hCG. This study and our recent report on inhibitory action of low doses of forskolin, 10(-12)-10(-9) M (Khanum, A., and Dufau, M.L. (1986) J. Biol. Chem. 261, 11456-11459) are indicative of a pertussis toxin-sensitive subunit of adenylate cyclase available for acute regulation of Leydig cell function. 8-bromo-cAMP bypasses the inhibitory effect of forskolin as well as AII. We have, therefore, demonstrated functional AII high-affinity receptor and an acute inhibitory effect of AII on hCG action in Leydig cells. Our results have provided evidence for a pertussis toxin-sensitive guanine nucleotide inhibitory protein as mediator of the effect of AII. These findings further emphasized the importance of the cAMP pathway in the Leydig cells, and studies also suggest that tubular and locally produced AII could negatively modulate luteinizing hormone stimulation of Leydig cells.

Inhibitory action of forskolin on adenylate cyclase activity and cyclic AMP generation.

In testicular Leydig cells, forskolin causes the expected stimulation of cAMP and testosterone production and potentiates gonadotropin-induced responses, when present in concentrations of 1-10 microM. In addition, when added at lower doses that did not affect cAMP generation and testosterone responses (100 nM), forskolin caused an increase in sensitivity to hormonal stimulation for all cAMP pools (extracellular, intracellular, and receptor-bound) and a 70% reduction in the ED50 for human chorionic gonadotropin (hCG) stimulation of testosterone production. Forskolin-induced increases in receptor-bound cAMP were less effective than those elicited by hCG in stimulating steroidogenesis. In contrast to the well-known stimulatory actions of forskolin, low doses of the diterpene (in the picomolar to nanomolar range) markedly inhibited the production of cAMP and testosterone. Such inhibitory actions of low-dose forskolin were prevented by preincubation of Leydig cells with pertussis toxin before addition of forskolin and/or hCG. Low concentrations of forskolin also inhibited adenylate cyclase activation by GTP and luteinizing hormone, and this effect was prevented by pretreatment of cell membranes with pertussis toxin. These studies have defined the stimulatory effects of forskolin on Leydig-cell cAMP pools, including potentiation of the hormonal increase in receptor-bound cyclic AMP by forskolin, and have provided additional evidence for the functional importance of cAMP compartmentalization during hormonal stimulation of steroidogenesis. We have also demonstrated a novel, high-affinity inhibitory action of forskolin upon adenylate cyclase activity and cyclic AMP generation, an effect that appears to be mediated by the Ni guanine nucleotide regulatory subunit of adenylate cyclase.

Effect of anticalmodulin drugs on testosterone synthesis in hCG stimulated mouse Leydig cells.

The effect of three anticalmodulin drugs, prepared in this laboratory and a commercially available drug Mastoparan, was tested on the secretion (or synthesis) of testosterone in hCG stimulated Leydig cells. The results of the use of drugs RN-IV A, RN-IV B and RN-IV C indicated that hCG (10 ng/ml), DbcAMP (0.1 mM) and cholera toxin (2 micrograms/ml)-stimulated testosterone production was inhibited in Leydig cells in a dose dependent manner. In hCG stimulated cells, the ID50 for drug RN-IV A was 2 microM, for drug RN-IV B was 25 microM and for drug RN-IV C was 130 microM. Based on ID50 the most effective drug was RN-IV A. Maximum inhibition of testosterone production was obtained at a concentration of 20 microM for drug RN-IV A, 150 microM for drug RN-IV B and 200 microM for drug RN-IV C. Further extensive experiments with drug RN-IV B showed that (a) at 100 microM concentration the drug does not impair the binding of receptor with 125I hCG, (b) the cAMP accumulation was prevented in a dose dependent manner reaching a minimal of 1.1 pM at 50 microM, compared with 3.5 pM in hCG (10 ng/ml) stimulated cells. The drug RN-IV B at a concentration of 100 microM, which failed to prevent conversion of exogenous pregnenolone or progesterone to testosterone, otherwise caused complete inhibition of testosterone production in hCG stimulated cells. Mastoparan also inhibited testosterone production in hCG stimulated cells in a dose dependent manner.(ABSTRACT TRUNCATED AT 250 WORDS)

Multistep regulation of Leydig cell function.

LH controls Leydig cell steroidogenesis by interaction with specific membrane receptors initiating membrane coupling events. Stimulation of the androgen pathways occurs mainly through cAMP mediated mechanism including LH induced guanyl nucleotide binding, membrane phosphorylation and adenylate cyclase activation. cAMP dependent kinase activation presumably causes phosphorylation of key proteins of the steroidogenic pathway and consequent increase in testosterone production. The hormone also appears to facilitate the androgen stimulus by a cyclic AMP independent mechanism located at the plasma membrane or intracellular sites. The stimulatory event can be negatively influenced by the action of certain peptide hormones (i.e. angiotensin II) through the guanyl nucleotide inhibitory subunit of adenylate cyclase (Gi). In recent studies we have presented evidence for a Ca2+ sensitive kinase system present in purified cell membranes. Gpp(NH)p, GTP, and phospholipid in presence of nanomolar Ca2+ induce phosphate incorporation into Mr 44,500 substrate with marked inhibition at microM Ca2+. Similarly a biphasic pattern of activation was observed with adenylate cyclase activity. Membrane phosphorylation may be a modifier of LH-stimulated adenylate cyclase activity and possibly other LH induced actions in the activated Leydig cell membrane. Furthermore we have defined the stimulatory effects of forskolin on all Leydig cell cyclic AMP pools and have provided additional evidence of functional compartmentalization and/or cAMP independent facilitory stimulus of steroidogenesis by the trophic hormone. The demonstration of a novel high affinity inhibitory action of forskolin upon adenylate cyclase activity and cyclic AMP generation mediated by the Gi subunit of adenylate cyclase has provided a new approach for direct evaluation of functional inhibitory influence of Gi subunit in the Leydig cell. The cultured fetal Leydig cell system has provided a useful model to elucidate mechanisms involved in the development of gonadotropin induced estradiol mediated desensitization of steroidogenesis. We have isolated from the fetal testis a small population (2-5% of total) of transitional cells with morphological characteristics of cells found in 15 day postnatal testis but functional capabilities of the adult cell. We have also demonstrated after appropriate treatment (i.e. estrogen, and frequent or a high gonadotropin dose) the emergence of a functional adult-like cell type from the fetal Leydig cell population.

C-terminal truncation of the neurokinin-2 receptor causes enhanced and sustained agonist-induced signaling. Role of receptor phosphorylation in signal attenuation.

The G protein-linked receptor for neurokinin A (NKA) couples to stimulation of phospholipase C and, in some cells, adenylyl cyclase. We have examined the function of the C-terminal cytoplasmic domain in receptor signaling and desensitization. We constructed C-terminal deletion mutants of the human NK-2 receptor (epitope tagged) to remove potential Ser/Thr phosphorylation sites, and expressed them in both mammalian and insect cells. When activated, truncated receptors mediate stronger and more prolonged phosphoinositide hydrolysis than wild-type receptor; however, the amplitude and kinetics of the NKA-induced rise in cytosolic Ca2+ remain unaltered. Protein kinase C (PKC)-activating phorbol ester abolishes wild-type receptor signaling but not mutant receptor signaling. Mutant receptors also mediate enhanced and prolonged cAMP generation, at least in part via PKC activation. When expressed in COS cells or Sf9 insect cells, the wild-type receptor is phosphorylated; receptor phosphorylation increases after addition of either NKA or phorbol ester. In contrast, mutant receptors are not phosphorylated by either treatment. Our results suggest that C-terminal Ser/Thr phosphorylation sites in the NK-2 receptor have a critical role in both homologous and heterologous desensitization. Removal of these phosphorylation sites results in a receptor that mediates sustained activation of signaling pathways and is insensitive to inhibition by PKC.