Corticotropin-releasing factor 1 receptor-mediated mechanisms inhibit colonic hypersensitivity in rats.

The potential relationship between stress and irritable bowel syndrome (IBS) symptomatology suggests a possible role for stress-mediating hormones, such as corticotropin-releasing factor (CRF), in the altered perception of stimuli in IBS patients. In previous studies, Wistar-Kyoto (WKY) rats with genetic indices of high anxiety demonstrated colonic hypersensitivity coupled with a high basal level of CRF within the central nervous system. In the current study we tested the hypothesis that a selective, non-peptide CRF1 receptor antagonist, antalarmin, would inhibit hypersensitivity in the WKY rat colon. Colonic sensitivity was determined by monitoring a visceromotor behavioural response during innocuous levels of colorectal distention (30 mmHg). In high anxiety WKY rats we found that antalarmin (20 mg kg-1, i.p.) significantly decreased the visceromotor response induced by colorectal distention. In a second study central administration (i.c.v.) of CRF was used to induce colonic hypersensitivity in lower anxiety Fischer 344 (F-344) rats, and in this model, antalarmin significantly inhibited the CRF-induced colonic hypersensitivity. In summary, a selective CRF1 receptor antagonist, antalarmin, inhibits colonic hypersensitivity apparent in WKY rats or in F-344 rats given a central administration of CRF. Our findings suggest that CRF1 receptor antagonism may represent a novel therapeutic approach for the treatment of IBS.

Anatomical distribution and cellular basis for high levels of aromatase activity in the brain of teleost fish: aromatase enzyme and mRNA expression identify glia as source.

Although teleost fish have higher levels of brain aromatase activity than any other vertebrate group, its function remains speculative, and no study has identified its cellular basis. A previous study determined aromatase activity in a vocal fish, the plainfin midshipman (Porichthys notatus), and found highest levels in the telencephalon and lower levels in the sonic hindbrain, which was dimorphic between and within (males) sexes. We have now localized aromatase-containing cells in the midshipman brain both by immunocytochemistry using teleost-specific aromatase antibodies and by in situ hybridization using midshipman-specific aromatase probes. Aromatase-immuno-reactivity and mRNA hybridization signal are consistent with relative levels of aromatase activity in different brain regions: concentrated in the dimorphic sonic motor nucleus, in a band just beneath the periaqueductal gray in the midbrain, in ventricular regions in the hypothalamus, and highest levels in the telencephalon especially in preoptic and ventricular areas. Surprisingly, double-label immunofluorescence does not show aromatase-immunoreactive colocalization in neurons, but instead in radial glia throughout the brain. This is the first study to identify aromatase expression mostly, if not entirely, in glial cells under normal rather than brain injury-dependent conditions. The abundance of aromatase in teleosts may represent an adaptation linked to continual neurogenesis that is known to occur throughout an individual's lifetime among fishes. The localization of aromatase within the intersexually and intrasexually dimorphic vocal-motor circuit further implies a function in the expression of alternative male reproductive phenotypes and, more generally, the development of natural, individual variation of specific brain nuclei.

Blunted stress cortisol response in abstinent alcoholic and polysubstance-abusing men.

BACKGROUND: This study tested cortisol responses to a psychological stressor in controls (CT) versus patients who were diagnosed as alcohol dependent (AD) or alcohol and stimulant dependent (ADSD) by DSM-IV criteria and who were abstinent for 3 to 4 weeks from alcohol and illicit drugs. Alcohol increases cortisol secretion acutely and during withdrawal. However, there is little information about abnormalities of hypothalamic-pituitary-adrenocortical (HPA) reactivity in recovering alcoholics. METHODS: Accordingly, we tested HPA function in the laboratory between 7:00 and 9:30 AM on control versus stress days. Stress consisted of a 20-min public speaking challenge with preparation and delivery of two short speeches, ostensibly evaluated for quality of delivery, whereas control involved relaxing for the same period. Cortisol was measured in saliva collected at baseline, stress or control, and recovery period, and also at home at 9:00 PM on one of the two days. RESULTS: The three groups did not differ in diurnal patterns of cortisol secretion on the rest day and 9:00 PM sample, which indicated that AD and ADSD patients had intact diurnal HPA regulation at rest. During speech stress, the CT subjects showed the expected cortisol increase (p < 0.0001), whereas neither AD nor ADSD patients responded significantly. Cortisol values were not accounted for by covariates such as depression, posttraumatic stress disorder, glucose metabolism, or anthropometric or demographic characteristics. CONCLUSIONS: The apparent stress hyporesponsiveness of the AD and ADSD patients suggests a persistent disruption of HPA function, perhaps due to incomplete recovery from prior abuse, or to a preexisting alteration in neural systems that regulate HPA responses to stress.

Evidence for visceral hypersensitivity in high-anxiety rats.

In patients with irritable bowel syndrome, anxiety is often associated with visceral pain. Based on this information we hypothesized that rats genetically predisposed to anxiety have an increased visceral sensitivity. To test this hypothesis, visceromotor reflex recordings in response to colorectal distention were used to estimate the level of visceral stimulation in high; moderate-, and low-anxiety rats. We compared the effect of innocuous colorectal distension in rats with and without sensitized colons. In nonsensitized rats visceromotor responses were increased by colorectal distention with the greatest response in the high-anxiety Wistar-Kyoto strain. Sensitization of the colon significantly increased visceromotor responses to colorectal distention in all rat strains. In summary, our data suggested that a manifestation of a genetically determined anxiety level appeared to be abnormal neural responsiveness of the gastrointestinal tract leading to visceral hypersensitivity in high-anxiety animals.

Corticosterone delivery to the amygdala increases corticotropin-releasing factor mRNA in the central amygdaloid nucleus and anxiety-like behavior.

The present study examined the effects of stereotaxic delivery of corticosterone to the amygdala on anxiety-like behavior and corticotropin-releasing factor (CRF) mRNA level in the central nucleus of the amygdala (CeA). Micropellets (30 microg) of crystalline corticosterone or cholesterol (control) were implanted bilaterally at the dorsal margin of the CeA in Wistar rats. Seven days post-implantation, anxiety-like behavior was accessed using an elevated plus-maze. CRF mRNA level in the CeA was determined by in situ hybridization 4 h after being tested on the elevated plus-maze. Corticosterone implants increased indices of anxiety on the elevated plus-maze and produced a concomitant increase in both basal level of CRF mRNA per neuron and the number of neurons with CRF hybridization signal in the CeA. The plus-maze increased CRF mRNA levels in the CeA of cholesterol implanted rats to the elevated basal levels observed in corticosterone treated animals. Exposure to the plus-maze did not increase CRF mRNA level in the CeA of corticosterone implanted rats beyond elevated basal levels. Taken together, these findings support the involvement of the amygdala in anxiety-like behaviors in response to chronically elevated corticosterone and suggests that elevated glucocorticoids may increase anxiety by inducing CRF expression in the CeA.

Ontogeny of angiotensin II type 1 receptor and cytochrome P450(c11) in the sheep adrenal gland.

In the present study we investigated the ontogeny of the expression of the type 1 angiotensin receptor (AT(1)R mRNA) and the zonal localization of AT(1)R immunoreactivity (AT(1)R-ir) and cytochrome P450(c11) (CYP11B-ir) in the sheep adrenal gland. In the adult sheep and in the fetus from as early as 90 days gestation, intense AT(1)R-ir was observed predominantly in the zona glomerulosa and to a lesser extent in the zona fasciculata, and it was not detectable in the adrenal medulla. AT(1)R mRNA decreased 4-fold between 105 days and 120 days, whereas AT(1)R mRNA levels remained relatively constant between 120 days and the newborn period. In contrast, both in the adult sheep and in the fetal sheep from as early as 90 days gestation, intense CYP11B-ir was consistently detected throughout the adrenal cortex and in steroidogenic cells that surround the central adrenal vein. In conclusion, we speculate that the presence of AT(1)R in the zona fasciculata, and the higher levels of expression of AT(1)R at around 100 days gestation, may suggest that suppression of CYP17 is mediated via AT(1)R at this time. The abundant expression of AT(1)R-ir and CYP11B-ir in the zona glomerulosa of the fetal sheep adrenal gland would also suggest that lack of angiotensin II stimulation of aldosterone secretion is not due to an absence of AT(1)R or CYP11B in the zona glomerulosa.

Regulation of proopiomelanocortin messenger ribonucleic acid levels in the ovine fetal anterior pituitary in vitro.

Parturition in sheep is dependent upon maturation of the fetal hypothalamo-pituitary-adrenocortical (HPA) axis. Anterior pituitary expression of the ACTH precursor, proopiomelanocortin (POMC), increases during the final days of gestation in spite of exponentially increasing fetal plasma cortisol levels. Lesion of the hypothalamic paraventricular nucleus prevents the late gestation increase in POMC mRNA. The purpose of this study was to examine glucocorticoid, corticotropin releasing factor (CRF) and arginine vasopressin (AVP) regulation of POMC mRNA levels in fetal anterior pituitary corticotropes in vitro and to address potential interactions between glucocorticoids and neuropeptides in regulating POMC. Anterior pituitaries from fetal sheep at two gestational ages (dGA; 118-125 dGA, n=9; 140-144 dGA, n=7) were enzymatically dispersed. POMC mRNA levels were determined at 24, 48 and 72 h post-dispersion. CRF, AVP and dexamethasone (DEX) regulation of POMC mRNA were determined at 24 and 72 h post-dispersion. The capacity of CRF and AVP to modulate DEX suppression of POMC mRNA levels was also examined. POMC mRNA was elevated at 24 h (P<0.01) and 48 h (P<0.05) post-dispersion compared to 0 h (immediately post-dispersion) in 140-144 dGA but not 118-125 dGA corticotropes. DEX suppressed POMC mRNA in a dose-dependent manner (when administered at 24 h post-dispersion) in the 140-144 dGA anterior pituitary cells but not 118-125 dGA anterior pituitary cells. Administration of DEX (10 nM) at 0 h prevented the increase in POMC mRNA levels observed at 24 h post dispersion in the 140-144 dGA group. Neither CRF nor AVP (administered at either 24 or 72 h post-dispersion) altered POMC mRNA levels in either 118-125 or 140-144 dGA anterior pituitary cells. Continuous exposure of anterior pituitary cells with either CRF or AVP (50 pM) through 96 h increased (P<0.05) POMC mRNA. No synergistic or additive effects were observed with CRF and AVP. Four hour pretreatment with CRF but not AVP (100 nM at 24 h post-dispersion) attenuated (P<0.05) DEX suppression of POMC mRNA levels in 140-144 dGA corticotropes. In conclusion, our results indicate that direct glucocorticoid suppression of POMC expression in fetal sheep initiates between approximately 120 and approximately 140 dGA, coincident with the period of gestation when fetal plasma cortisol is exponentially rising. Further, while short duration exposure of fetal corticotropes to either CRF or AVP had no effect on POMC mRNA, CRF appears capable of interfering with glucocorticoid suppression of POMC mRNA. The latter observation provides a potential mechanism via which the fetal PVN may counter rising fetal plasma cortisol concentrations resulting in the previously observed late gestation increase in anterior pituitary POMC mRNA.

Corticotropin-releasing factor receptor expression in the pituitary of fetal sheep after lesion of the hypothalamic paraventricular nucleus.

Both the capacity of CRF to release ACTH and the number of binding sites for CRF in the anterior pituitary decline during the final weeks of gestation in fetal sheep. The present study examined regulation of pituitary CRF receptor expression by the hypothalamic paraventricular nucleus (PVN) during late gestation in fetal sheep. Bilateral radiofrequency lesions of the PVN (PVN-Lx; n = 4) or sham lesions (SHAM; n = 5) were performed in fetal sheep at 118-122 days of gestational age (dGA). Pituitary glands from PVN-Lx and SHAM fetuses were collected at 139-142 dGA (term, approximately 148 dGA). Dual-label in situ hybridization was performed using a digoxigenin-labeled ovine POMC complementary RNA, together with a 35S-labeled ovine CRF type I (CRF1) receptor complementary RNA, to localize and quantify CRF1 receptor mRNA in POMC-hybridizing cells. Binding of [125I]-ovine CRF was also examined in the fetal pituitary of both PVN-Lx and SHAM fetuses using in situ autoradiography. The hybridization signal for the CRF1 receptor mRNA was primarily restricted to POMC-expressing cells in the anterior pituitary of both PVN-Lx and SHAM fetuses; no hybridization signal for the CRF1 receptor was observed in the neurointermediate lobe (NIL) in either group. The hybridization signal for CRF1 receptor mRNA in anterior pituitary corticotropes of PVN-Lx fetuses was significantly lower in both the inferior and superior regions of the anterior pituitary, compared with SHAM fetuses (P < 0.05). In the inferior region of the anterior pituitary, the percentage of POMC-hybridizing cells containing CRF1 receptor hybridization signal was significantly greater in PVN-Lx (90+/-7%; mean +/- SEM), compared with SHAM (67+/-6%; P < 0.05) fetuses. No differences in the percentage of POMC cells containing CRF1 receptor hybridization signal were observed in the superior region of the anterior pituitary between PVN-Lx (89+/-8%) and SHAM (87+/-9%). Binding of [125I]-ovine CRF (oCRF) was significantly greater in anterior pituitaries of PVN-Lx (140+/-19 mean arbitrary densitometry U +/- SEM), compared with SHAM (73+/-23; P < 0.05) fetuses. For both PVN-Lx and SHAM fetuses, there were no differences within group in [125I]-oCRF binding between the inferior and superior regions of the anterior pituitary. A weak, but significant (P < 0.05), autoradiographic signal for [125I]-oCRF binding was observed in the NIL of both SHAM and PVN-Lx fetal sheep. The level of [125I]-oCRF binding was significantly lower in the NIL, compared with anterior pituitary, for both SHAM (P < 0.01) and PVN-Lx fetuses. There were no differences in [125I]-oCRF binding in the NIL between SHAM and PVN-Lx fetal sheep. Our findings support a role for the PVN in regulating anterior pituitary CRF1 receptor expression in the late-gestation sheep fetus.

Structure and function of the ovine type 1 corticotropin releasing factor receptor (CRF1) and a carboxyl-terminal variant.

Corticotropin releasing factor (CRF) is the major neuropeptide regulating the hypothalamo-pituitary-adrenocortical axis in most species. A pituitary receptor for CRF (designated CRF1) belonging to the seven-transmembrane helix, G-protein-coupled receptor superfamily has been cloned for human, rat, mouse and xenopus. Since ovine CRF shares only 84% identity to human/rat CRF (h/rCRF) we postulated that the sheep pituitary CRF1 receptor may have similarly diverged from the rodent and human CRF1. We report the molecular cloning of an ovine pituitary cDNA containing a 1245 bp open reading frame encoding a 415 amino acid sheep CRF1 receptor 78, 86, 94, and 95% homologous to xenopus, chicken, rat, mouse, and human CRF1, respectively. The divergence in primary structure between the sheep CRF1 and the other mammalian CRF1s is primarily localized to the extracellular amino terminal domain of the receptor (18 of 22 divergent residues, ovine vs human CRF1). A variant of the oCRF1 was also isolated (oCRF1var) with 133 bp deleted from nucleotide (nt) 1080 to nt 1213 of the open reading frame (ORF) resulting in a new ORF of 1176 nt predicting a 392 residue CRF1 variant receptor. The 133 bp deletion would cause a frame-shift at residue 358 within the carboxyl-third of the seventh transmembrane domain (TM7) resulting in a shortened cytoplasmic tail with a new amino acid sequence from residue 358 to 392. Scatchard analysis of saturation curves using membrane prepared from Cos 7 cells transfected with oCRF1 or oCRF1var indicated that both wild-type and variant receptors were expressed similarly (number of CRF binding sites) and both bound oCRF with high affinity [oCRF1 (Kd): 2.5 + 1.6 nM; oCRF1var: 5.1 + 2.3 nM]. The non-hydrolyzable GTP analogue (GTPgammaS) lowered the affinity of both wild-type and variant oCRF1 receptors to a similar extent (oCRF1: 18.2 nM; oCRF1var: 22.4 nM). Both wild-type and variant oCRF1 receptors exhibited approximately 10-fold greater selectivity for oCRF and sauvagine compared to h/rCRF or alpha-helical [9-41]oCRF. CRF effectively stimulated the accumulation of cAMP (EC50 = 51 pM) in Cos 7 cells transiently transfected with wild-type but not variant oCRF1 receptor. In Cos 7 cells transfected with oCRF1var, cAMP accumulation was only observed at the highest concentration of oCRF utilized (100 nM). Basal (unstimulated) levels of cAMP in Cos 7 cells transfected with oCRF1var (in the presence of 2 mM IBMX) were approximately 50% lower than for the wild-type oCRF1. Differences in cAMP accumulation could not be attributed to differences in receptor number since total binding sites in the transfected cells were not different between wild-type or variant oCRF1 receptors. Agonist-induced receptor internalization, determined as the percent of total [125I] Tyr0-oCRF bound located in the acid-resistant fraction of transfected Cos 7 cells, increased with time (0-60 min at 37 degrees C) for both wild-type and variant oCRF1. Wild-type CRF1 internalized approximately 2-fold greater percent of total [125I] Tyr0-oCRF bound compared to the variant receptor. In summary, an ovine CRF1 and a CRF1 cytoplasmic tail receptor variant displaying high affinity binding to oCRF as well as selectivity for oCRF vs h/rCRF, were cloned from an adult sheep pituitary cDNA library. GTPgammaS studies indicate that both variant and wild-type receptors couple efficiently to Galphas however, only the wild-type oCRF1 is capable of stimulating cAMP production at physiological levels of CRF. Agonist-induced internalization of the ovine CRF1var is also reduced compared to the wild-type CRF1 receptor. We suggest that the oCRF1var interacts efficiently with Galphas but is unable (post-hormonal binding) to effectively stimulate G-protein activation of adenylate cyclase, indicating that the cytoplasmic tail of the CRF1 can modulate receptor function related to signal transduction. (ABSTRACT TRUNCATED)

Expression of proopiomelanocortin and prohormone convertase-1 and -2 in the late gestation fetal sheep pituitary.

The biological activity of fetal plasma immunoreactive ACTH has been reported to increase during the final weeks of gestation in fetal sheep, indicative of enhanced processing of POMC to ACTH. The present study was aimed at examining the expression and localization of the prohormone convertases, PC1 and PC2, in the pituitary of fetal sheep during the final weeks of gestation. Pituitaries were obtained from fetal sheep during the final 50 days gestation (dGA) at 100-107 dGA (n = 6), 117-121 dGA (n = 6), 126-130 dGA (n = 7), and 144-147 dGA (n = 8; term = approximately 148 dGA). Pituitaries were cryosectioned and subjected to dual labeling in situ hybridization using 35S-labeled PC1 and/or PC2 complementary RNA probes with a digoxigenin-labeled POMC complementary RNA to localize and quantify PC1 and PC2 messenger RNA (mRNA) in POMC-hybridizing cells. Immunocytochemistry was also performed to assess coexpression of PC1 and PC2 with ACTH in the fetal pituitary. PC1 mRNA was heterogeneously distributed in the anterior pituitary (AP) at all gestational ages examined, with hybridization signals observed over POMC-expressing cells (corticotropes) as well as over noncorticotrope phenotypes. The inferior region of the AP contained an approximately 3-fold greater (P < 0.01) percentage of POMC cells containing PC1 transcripts compared with the superior region of the AP. The proportion of POMC cells containing PC1 was significantly higher (P < 0.01) in the 100-107 dGA and 144-147 dGA groups than in the 117-121 dGA and 126-130 dGA groups in both inferior and superior AP. The intensity of the PC1 hybridization signal over POMC-expressing cells was also about 2- to 4-fold greater (P < 0.01) in the inferior compared with the superior region of the fetal AP; the intensity of the PC1 hybridization signal associated with POMC cells remained constant within the AP region and did not change over the gestational ages examined. Hybridization for PC1 was highly variable over regions of AP not hybridizing for POMC, probably due to differences in the level of mRNA for PC1 between phenotypes. Similar to POMC cells, the average hybridization signal for PC1 over non-POMC-hybridizing regions was about 2-fold greater in the inferior vs. superior AP. A weak PC2 hybridization signal was observed over a small number of unidentified phenotypes in the fetal AP at all ages examined; no POMC cells were found to contain PC2 hybridization signal. In the neurointermediate lobe, POMC, PC1, and PC2 were ubiquitously expressed at all ages. Levels of PC1 and PC2 mRNA in the fetal neurointermediate lobe did not change over the period of gestation examined. Immunocytochemical analysis of PC1 and PC2 with ACTH confirmed the pattern of expression and the extent of coexpression observed with in situ hybridization methods. We conclude that both PC1 and PC2 are likely to contribute to POMC processing in the fetal pituitary during the final weeks of gestation.

The incidence of lower-pole nephrolithiasis--increasing or not?

OBJECTIVE: To determine if the incidence of lower-pole nephrolithiasis is increasing. METHODS: A previously published meta-analysis of trends in the location of stones in the kidney, using data from 1984 to 1992, determined the percentage of lower pole stones in 26,722 kidney stones treated by extracorporeal shockwave lithotripsy (ESWL). We performed prospective studies on all patients treated by ESWL for a single renal stone (not manipulated from the ureter) in two organizations: at Lithotripters Inc., 47,303 stones were treated with ESWL by 1000 urologists in private practice from 1989 to 1995. At the Midwest Urologic Stone Unit, 9357 stones were treated with ESWL by 200 urologists in private practice from 1987 to 1995. The distribution of stones in both samples was compared with that reported earlier. RESULTS: The meta-analysis for stone location trends from the previously published article suggested that the percentage of kidney stones in the lower pole at ESWL increased erratically from 1984 to 1989 but was then stable for 3 years. The Lithotripters Inc. sample showed an essentially constant incidence from 28% in 1990 to 30% in 1995, and the Midwest Urologic Stone Unit sample showed an essentially constant incidence from 35% in 1988 to 36% in 1995. CONCLUSION: The incidence of lower pole nephrolithiasis has remained stable from 1990.

Fetal sheep pituitary proopiomelanocortin in late gestation: effect of bilateral lesions of the paraventricular nucleus on regional and cellular messenger ribonucleic acid levels.

Previous experiments have clearly indicated that the successful completion of ovine gestation is dependent upon fetal adrenocortical maturation and the associated preterm rise in fetal plasma cortisol. The purposes of this study were to: 1) examine pituitary POMC messenger RNA (mRNA) levels during normal fetal development; and 2) examine the effects of bilateral lesion of the fetal paraventricular nucleus (PVN) on levels and spatial distribution of pituitary POMC mRNA. Pituitary glands were collected from intact fetal sheep of four gestational ages [100-107 days gestational age (dga), n = 8; 117-121 dga, n = 9; 126-130 dga, n = 9; 144-147 dga, n = 8]. Lesions of the PVN (PVN Lx; n = 4) or sham lesions (Sham; n = 5) were performed at 118-122 dga. Pituitary glands from PVN Lx and Sham fetuses were collected at 139-142 dga (term approximately 147 dga). POMC mRNA levels were determined by in situ hybridization. POMC transcript levels were determined by both regional analysis (20x magnification) and analysis of individual corticotropes (400x magnification). There was no difference among gestational age groups in superior anterior pituitary (AP) POMC mRNA levels determined by regional or cellular analysis. POMC mRNA levels were significantly greater in the inferior AP at 144-147 dga, compared with other gestational ages, using regional analysis (P = 0.003) or analysis of individual corticotropes (P < 0.01). POMC mRNA levels in the neurointermediate lobe in 126- to 130-dga fetuses were significantly greater than those in younger fetuses (P = 0.005) but not those in 144- to 147-dga fetuses. There was no difference in POMC mRNA levels in the superior AP between PVN Lx and Sham, using regional analysis or analysis of individual corticotropes. In the inferior AP, there was a significant decrease in POMC mRNA levels in PVN Lx, compared with Sham, using both regional analysis (P < 0.01) and cellular analysis (P < 0.01). There was no difference in POMC mRNA levels in the neurointermediate lobe as the result of bilateral PVN Lx. Our findings support that basal AP POMC mRNA levels are heterogenously distributed in the ovine fetal AP, with POMC mRNA levels in the inferior AP being significantly greater than in superior AP, by 144-147 dga. We further found that the higher POMC mRNA levels in the inferior AP reflect significantly higher corticotrope POMC transcripts and not simply a greater density of corticotropes in this AP region. The increase in POMC mRNA levels at 144-147 dga in the inferior AP seems unrelated to the onset of adrenocortical maturation (at approximately 125-130 dga). Finally, we report that increase in corticotrope POMC transcripts during late gestation in the inferior AP requires an intact PVN.

Modification of gonadotropin releasing hormone (GnRH) mRNA expression in the retinal-recipient Thalamus.

Although the environmental cues that trigger reproductive behaviors are known for many species, the mechanisms through which these signals influence the neurochemistry of the brain to produce behavior have been elusive. In this study, we describe a retinally modulated system of gonadotropin releasing hormone (GnRH) producing neurons in the thalamus of the plainfin midshipman fish, Porichthys notatus. Previously, we cloned and sequenced the cDNA for prepro-GnRH in midshipman. Here, using in situ hybridization, we localized prepro-GnRH mRNA to the ventrolateral nucleus of the thalamus, three divisions of the preoptic area, the ganglion of the terminal nerve, and the olfactory bulb. Since the thalamus, terminal nerve ganglion, and preoptic area have been associated with visual functions, we investigated the retinal connections in midshipman. In particular, biocytin tract tracing delineated a reciprocal connection between the ventrolateral nucleus of the thalamus and the retina. Retinofugal projections are exclusively contralateral. Experimental manipulation of this retinalthalamic loop through complete optic nerve transection shows that GnRH mRNA expression in the contralateral ventrolateral nucleus may be influenced by the retina. We hypothesize that a reciprocal retinothalamic GnRH circuit is important in modulating the expression of seasonal reproductive behaviors.

Structure, localization, and molecular phylogeny of a GnRH cDNA from a paracanthopterygian fish, the plainfin midshipman (Porichthys notatus).

Multiple forms of gonadotropin releasing hormone (GnRH) are found within several species of teleost fishes. Within the infradivision Euteleostei, the superorder Paracanthopterygii represents one of the last major groups to be examined with respect to the GnRH mRNA sequence. The plainfin midshipman, Porichthys notatus, is a common member of this superorder which is intermediate between the ancestral euteleost taxa and the more derived Acanthopterygians (percomorphs). The goals of this study were to: (1) determine the cDNA sequence of prepro-GnRH in the plainfin midshipman, (2) address the anatomical localization of midshipman prepro-GnRH gene expression, and (3) perform a cladistic analysis using all currently known cDNA sequences of prepro-GnRH. We report 460 base pair of cDNA sequence containing the entire protein coding region, and 5'- and 3'-untranslated regions. The deduced amino acid sequence indicates that this cDNA encodes a GnRH decapeptide identical in sequence to that originally isolated in salmon (Trp7, leu8 - GnRH). Northern analysis demonstrated transcripts in brain, ovary, and testis (600-700 nucleotides). PCR showed that the ovarian prepro-GnRH was identical to that found in brain. In situ hybridization labeled neurons in the ganglion of the terminal nerve and the preoptic area, forebrain areas previously observed to contain GnRH-like immunoreactivity. Last, a phylogenetic analysis of 18 prepro-GnRH sequences grouped the Paracanthopterygii with the Acanthopterygii. However, this recent clade was distinct from two separate and more ancestral lineages, the Paracanthopterygii (salmonids) and Ostariophysi (represented by catfish).

Prostaglandin synthase activity of fetal sheep cotyledons at 122 days of gestation and term: expression of prostaglandin synthetic capacity in fetal cotyledonary tissue near labor is location-dependent.

An activity assay is described quantification of prostaglandin synthase (PGHS) in sheep placental cotyledon under initial velocity conditions through measurement of the stable product prostaglandin E2 (PGE2). The effects of temperature, time, and substrate concentration on initial reaction velocity, and lipoxygenase and PGHS product formation in cotyledonary tissue, were examined in detail. We used this activity assay to determine whether or not an increase of active PGHS by placental location within the uterus might contribute selected prostaglandins (PG) for the directed initiation of parturition. Sheep cotyledon tissue was collected (n = 6 animals) from the ventral aspect of the uterine body, mid-horn, and horn tip at 122 days of gestation (dga), and from the same locations in the ventral body and horn tip at 142-145 dga (in animals at term but not in labor; n = 4). At 122 dga, there was no increase in active PGHS in cotyledonary tissue from the horn tip, mid-horn, or uterine body. By 142-145 dga, the horn showed significantly (p < 0.01) more enzyme activity than the body. At the same time, production of PGE2, expressed as a percentage of total eicosanoids, had not changed significantly. The development of an increase in PGHS toward the uterine tip implies that variations in regional PG production may contribute to the progression of labor.

Changes in estrogen receptor messenger ribonucleic acid in sheep fetal and maternal tissues during late gestation and labor.

OBJECTIVE: Our purpose was to investigate whether there is an increase in messenger ribonucleic acid for estrogen receptor in critical maternal or fetal tissues in the last third of pregnancy and during labor in sheep. STUDY DESIGN: Estrogen receptor messenger ribonucleic acid was measured by Northern hybridization analysis in fetal-placental and maternal uterine tissues during the last third of pregnancy and during spontaneous or cortisol-induced labor in sheep. Statistical differences were assessed with two-way analysis of variance. RESULTS: No estrogen receptor messenger ribonucleic acid was observed in amnion or chorion in any animal studied. There were no gestational age related changes in estrogen receptor messenger ribonucleic acid in any tissues between 100 and 145 days' gestation. During spontaneous and cortisol-induced labor estrogen receptor messenger ribonucleic acid increased significantly (p < 0.05) in myometrium, endometrium, and cervix. No increase was observed in the fetal placental cotyledon and mesometrium. Estrogen receptor messenger ribonucleic acid was markedly decreased (p < 0.05) in myometrium and endometrium after fetal adrenalectomy. CONCLUSION: An increase in estrogen receptor messenger ribonucleic acid in association with labor may contribute part of the mechanism by which estrogens exert their influence on the process of parturition.

Pediatric low energy lithotripsy with the Lithostar.

Between November 1988 and July 1993, 238 renal stones and 208 ureteral stones were treated in 446 pediatric patients using 26 mobile and 2 fixed base Siemens Lithostar lithotriptors. The stones were treated by a group of 245 urologists using the modified Puigvert technique and the standard shock tube. The success rate for renal stones (asymptomatic fragments less than 4 mm.) was 76.6%, stone-free rate was 67.9%, retreatment rate was 14.1% and ancillary procedures were performed in 36.3%. The stone-free rate for ureteral stones was 91.1%, retreatment rate was 3.5% and ancillary procedures were performed in 17.7%. Anesthesia was required in 31% of the renal and 21% of the ureteral procedures. Sepsis in a 6-year-old child after treatment of a ureteral stone was the only major complication. Low energy lithotripsy with the Lithostar in our series of pediatric patients was safe and effective.

Effects of stents on lithotripsy of ureteral calculi: treatment results with 18,825 calculi using the Lithostar lithotriptor.

Between November 14, 1988 and August 1, 1993, 18,825 ureteral calculi were treated in the United States using 25 different mobile and 2 fixed base Siemens Lithostar lithotriptors. Lithotripsy was performed by 1,012 urologists using the modified Puigvert technique. The overall stone-free rate was 83.8% with a retreatment rate of 10.8%. The stone-free rate varied from 85.8% with stones of 10 mm. or smaller to 67.9% for stones larger than 20 mm. A ureteral stent or catheter was placed before lithotripsy in 19.3% of all treatments and 80.7% had in situ treatment without instrumentation. For calculi of any size, the use of ureteral stents or catheters had no effect on treatment outcome at any ureteral location.

Low energy lithotripsy with the Lithostar: treatment results with 19,962 renal and ureteral calculi.

Between November 1988 and January 1992, 19,962 renal and ureteral calculi were treated in the United States using 18 different mobile and 2 fixed base Lithostar lithotriptors. Lithotripsy was performed on 11,516 renal and 8,446 ureteral calculi by 750 urologists using the same technique. The success rate (asymptomatic with stone fragments of 4 mm. or less) for renal stones was 87.9%, the stone-free rate was 68.9% and the retreatment rate was 16.5%. Auxiliary procedures were performed in 32.2% of the renal calculi. The success rate for ureteral calculi was 89.5%, the stone-free rate was 83.5% and the retreatment rate was 10.7%. Auxiliary procedures were performed in 25.5% of the ureteral calculi. The overall success rate was 88.4% stone-free rate 75.5%, retreatment rate 14.0% and auxiliary procedure rate 29.4%. Anesthesia personnel were used in 1.9% of the cases. Low energy extracorporeal shock wave lithotripsy was found to be safe and effective.