A biopsy-integrated algorithm for determining Gleason 6 upgrading risk stratifies risk of active surveillance failure in prostate cancer.

INTRODUCTION: A significant proportion of patients that fail active surveillance (AS) for prostate cancer management do so because of cancer upgrading. A previously validated upgrading nomogram generates a score that predicts risk of biopsy Gleason 6 upgrading following radical prostatectomy in lower-risk populations that are candidates for Active Surveillance (Cancer, 2013). OBJECTIVES: We hypothesize that the upgrading risk (UR) score generated by this nomogram at diagnosis improves the ability to predict patients that will subsequently fail AS. METHODS: To evaluate the nomogram, retrospective data from several institutional cohorts of patients who met AS criteria, group 1 (n = 75) and group 2 (n = 1230), were independently examined. A UR score was generated using the coefficients from the nomogram consisting of PSA density (PSAD), BMI, maximum % core involvement (MCI), and number of positive cores. AS failure was defined as Gleason score (GS) >6, >50 % maximum core involvement, or >2 positive cores on biopsy. Univariate and multivariate Cox proportional-hazards regression models, upgrading risk score, and other clinicopathologic features were each assessed for their ability to predict AS failure. RESULTS: Clinicopathologic parameters were similar in both groups with the exception of mean PSAD (0.13 vs. 0.11, p < 0.01) and follow-up (2.1 vs. 3.2 years, p = 0.2). Most common cause of AS failure was GS > 6 (group 1) compared to >2 positive cores (group 2). On univariate analysis in both populations, features at diagnosis including PSAD and the UR score were significant in predicting AS failure by upgrading (Gleason > 6) and any failure. Multivariate analysis revealed the UR score predicts AS failure by GS upgrading (HR 1.8, 95 % CI 1.12-2.93; p = 0.01) and any failure criteria (HR 1.7, 95 % CI 1.06-2.65); p = 0.02) for group 1. Likewise, the UR score in group 2 predicts AS failure with GS upgrading (HR 1.3, 95 % CI 1.15-1.42; p < 0.0001) and any failure criteria (HR 1.18, 95 % CI 1.18-1.38; p < 0.0001). An ROC generated an AUC of 0.66. Decision curve analysis demonstrated a high net benefit for the UR score across a range of threshold probabilities. Based on these outcomes, at 3 years, patients in the lowest risk quartile have a 15 % risk of AS failure versus a 46 % risk in the highest quartile (p < 0.0001). CONCLUSIONS: The UR score was predictive of pathologic AS failure on multivariate analysis in several AS cohorts. It outperformed single clinicopathologic criteria and may provide a useful adjunct using clinicopathologic data to stratify patients considering AS.

Primary cutaneous adenosquamous carcinoma of the penis: the first characterization of HPV status in this rare and diagnostically challenging entity with review of glandular carcinomas of the penis.

Glandular and pseudoglandular tumors of the penile skin are extremely uncommon and can present diagnostic challenges. Primary adenosquamous carcinoma of the penis is an extremely rare tumor, composed of distinct areas of malignant squamous and glandular cells, making it a diagnostically challenging entity. The World Health Organization (WHO) recognizes several subtypes of squamous cell carcinoma (SCC), each with its own distinctive pathologic appearance, clinical associations and prognosis. Among these variants is the exceedingly uncommon adenosquamous carcinoma (ASC), representing 1%-2% of all SCC of the penis. Recent large studies have interrogated the presence of human papillomavirus (HPV) in malignant penile tumors and have shown specific morphologic patterns and clinical presentations to associate with HPV status. However, given the rarity of the adenosquamous variant of SCC, it has largely been excluded from these studies. The glandular components of these lesions can present a confusing appearance, particularly when a large tumor is represented on a small biopsy. Here we describe a difficult histologic presentation of this rare tumor, with the first published characterization of the HPV status of this subtype. This case represents a distinctly unusual case of metastatic HPV-positive primary cutaneous adenosquamous carcinoma of the penis.

Receptor-associated resistance to growth hormone-releasing factor in dwarf "little" mice.

Anterior pituitaries from the dwarf mouse strain "little" did not release growth hormone or accumulate adenosine 3',5'-monophosphate (cyclic AMP) in response to human and rat growth hormone-releasing factor (GRF). Dibutyryl cyclic AMP, as well as the adenylate cyclase stimulators forskolin and cholera toxin, markedly stimulated growth hormone (GH) release. The basis of the GH deficiency in the little mouse may therefore be a defect in an early stage of GRF-stimulated GH release related either to receptor binding or to the function of the hormone-receptor complex.

Decreased hypothalamic growth hormone-releasing hormone content and pituitary responsiveness in hypothyroidism.

The effects of thyroidectomy (Tx) and thyroxine replacement (T4Rx) on pituitary growth hormone (GH) secretion and hypothalamic GH-releasing hormone (GRH) concentration were compared to define the mechanism of hypothyroid-associated GH deficiency. Thyroidectomized rats exhibited a complete loss of pulsatile GH secretion with extensive reduction in GRH responsiveness and pituitary GH content. Cultured pituitary cells from Tx rats exhibited reduced GRH sensitivity, maximal GH responsiveness, and intracellular cyclic AMP accumulation to GRH, while somatostatin (SRIF) suppressive effects on GH secretion were increased. Hypothalamic GRH content was also markedly reduced. T4Rx completely restored hypothalamic GRH content and spontaneous GH secretion despite only partial recovery of pituitary GH content, GRH and SRIF sensitivity, and intracellular cyclic AMP response to GRH. The results indicate multiple effects of hypothyroidism on GH secretion and suggest that a critical role of T4 in maintaining normal GH secretion, in addition to restoring GH synthesis, is related to its effect on hypothalamic GRH.

Measurement of growth hormone-releasing hormone and somatostatin in hypothalamic-portal plasma of unanesthetized sheep. Spontaneous secretion and response to insulin-induced hypoglycemia.

To elucidate the role of growth hormone (GH)-releasing hormone (GRH) and somatostatin (SRIH) in the regulation of the growth hormone (GH) secretory pattern, we collected portal blood from five unanesthetized ovariectomized ewes for repeated measurements of GRH and SRIH simultaneous with those of peripheral GH. Hormones were measured at 10-min intervals for 5.5 h and their interrelationships analyzed. Mean portal GRH was 20.4 +/- 6.7 (SD) pg/ml and the estimated overall secretion rate was 13 pg/min. GRH secretion was pulsatile with peaks of 25-40 pg/ml and a mean pulse interval of 71 min. Mean portal SRIH was 72 +/- 33 pg/ml and the estimated overall secretion rate was 32 pg/min. SRIH secretion was also pulsatile with peaks of 65-160 pg/ml and a mean pulse interval of 54 min. The GH pulse interval was 62 min. A significant association was present between GRH and GH secretory peaks though not between GRH and SRIH or SRIH and GH. Insulin hypoglycemia resulted in a rapid and brief stimulation of SRIH secretion followed by a decline in GH levels. No effect was observed on GRH secretion until 90 min, when a slight increase occurred. The results suggest (a) the presence of an independent neural rhythmicity of GRH and SRIH secretion with a primary role of GRH in determining pulsatile GRH secretion, and (b) that the inhibitory effects of insulin hypoglycemia on GH in this species are attributable to a combination of enhanced SRIH secretion and possibly other factors, though without significant inhibition of GRH.

Dipeptidylpeptidase IV and trypsin-like enzymatic degradation of human growth hormone-releasing hormone in plasma.

The plasma enzyme responsible for primary proteolytic cleavage of growth hormone-releasing hormone (GRH) at the 2-3 amino acid bond was characterized. Native GRH[GRH(1-44)-NH2 and GRH(1-40)-OH], and COOH-terminally shortened fragments [GRH(1-32)-NH2 and GRH(1-29)-NH2] were rapidly cleaved, while GRH(2-32)-NH2 was not degraded at this site. Moreover, degradation to GRH(3-44)-NH2 was unaffected by an aminopeptidase inhibitor, indicating that this metabolite was generated from a single step cleavage by a dipeptidylpeptidase (DPP) rather than sequential aminopeptidase cleavages. Conversion to GRH(3-44)-NH2 was blocked by diprotin A, a DPP type IV (DPP IV) competitive inhibitor. D-Amino acid substitution at either position 1 or 2 also prevented hydrolysis, characteristic of DPP IV. Analysis of endogenous plasma GRH immunoreactivity from a human GRH transgenic pig revealed that the major peak coeluted with GRH(3-44)-NH2. Native GRH exhibited trypsin-like degradation at the 11-12 position but cleavage at the 12-13 site occurred only with GRH(1-32)-NH2 and GRH(1-29)-NH2. Formation of these metabolites was independent of prior DPP IV hydrolysis but was greatly reduced by trypsin inhibitors. Evaluation of plasma stability of potential GRH super analogues, designed to resist degradation by these enzymes, confirmed that GRH degradation in plasma occurs primarily by DPP IV, and to a lesser extent by trypsin-like enzyme(s).

Rapid enzymatic degradation of growth hormone-releasing hormone by plasma in vitro and in vivo to a biologically inactive product cleaved at the NH2 terminus.

The effect of plasma on degradation of human growth hormone-releasing hormone (GRH) was examined in vitro and in vivo using high performance liquid chromatography (HPLC), radioimmunoassay (RIA), and bioassay. When GRH(1-44)-NH2 was incubated with human plasma, the t1/2 of total GRH immunoreactivity was 63 min (RIA). However, HPLC revealed a more rapid disappearance (t1/2, 17 min) of GRH(1-44)-NH2 that was associated with the appearance of a less hydrophobic but relatively stable peptide that was fully immunoreactive. Sequence analysis indicated its structure to be GRH(3-44)-NH2. Identity was also confirmed by co-elution of purified and synthetic peptides on HPLC. Biologic activity of GRH(3-44)-NH2 was less than 10(-3) that of GRH(1-44)-NH2. After intravenous injection of GRH(1-44)-NH2 in normal subjects, a plasma immunoreactive peak with HPLC retention comparable to GRH(3-44)-NH2 was detected within 1 min and the t1/2 of GRH(1-44)-NH2 (HPLC) was 6.8 min. The results provide evidence for GRH inactivation by a plasma dipeptidylaminopeptidase that could limit its effect on the pituitary.

Multi-quadrant biopsy technique improves diagnostic ability in large heterogeneous renal masses. Abel EJ, Heckman JE, Hinshaw L, Best S, Lubner M, Jarrard DF, Downs TM, Nakada SY, Lee FT Jr, Huang W, Ziemlewicz T.J Urol. 2015 Oct;194(4):886-91. [Epub 2015 Mar 30]. doi: 10.1016/j.juro.2015.03.106.

PURPOSE: Percutaneous biopsy obtained from a single location is prone to sampling error in large heterogeneous renal masses, leading to nondiagnostic results or failure to detect poor prognostic features. We evaluated the accuracy of percutaneous biopsy for large renal masses using a modified multi-quadrant technique vs. a standard biopsy technique. MATERIALS AND METHODS: Clinical and pathological data for all patients with cT2 or greater renal masses who underwent percutaneous biopsy from 2009 to 2014 were reviewed. The multi-quadrant technique was defined as multiple core biopsies from at least 4 separate solid enhancing areas in the tumor. The incidence of nondiagnostic findings, sarcomatoid features and procedural complications was recorded, and concordance between biopsy specimens and nephrectomy pathology was compared. RESULTS: A total of 122 biopsies were performed for 117 tumors in 116 patients (46 using the standard biopsy technique and 76 using the multi-quadrant technique). Median tumor size was 10cm (IQR: 8-12). Biopsy was nondiagnostic in 5 of 46 (10.9%) standard and 0 of 76 (0%) multi-quadrant biopsies (P = 0.007). Renal cell carcinoma was identified in 96 of 115 (82.0%) tumors and nonrenal cell carcinoma tumors were identified in 21 (18.0%). One complication occurred using the standard biopsy technique and no complications were reported using the multi-quadrant technique. Sarcomatoid features were present in 23 of 96 (23.9%) large renal cell carcinomas studied. Sensitivity for identifying sarcomatoid features was higher using the multi-quadrant technique compared to the standard biopsy technique at 13 of 15 (86.7%) vs. 2 of 8 (25.0%) (P = 0.0062). CONCLUSIONS: The multi-quadrant percutaneous biopsy technique increases the ability to identify aggressive pathological features in large renal tumors and decreases nondiagnostic biopsy rates.

De novo calcification of liver and nodal metastases in prostate carcinoma.

Prostate cancer has a distinctly recognized pattern of metastases: multifocal and osteoblastic lesions involving the axial skeleton and non-calcified lymph nodes in the pelvic and lumbar aortic groups. Most adenocarcinomas are capable of producing macrocalcification. We report a case of prostate cancer with de novo calcified metastases to the liver and retroperitoneal lymph nodes mimicking the pattern usually seen in mucin-producing adenocarcinomas arising from the gastrointestinal tract. To our knowledge, this is the first such case to be reported in the literature. We propose a multifactorial mechanism that supports dystrophic calcification in this case. The knowledge of atypical presentation of metastatic disease can prevent diagnostic delay and prompt initiation of therapy.

Pursuing Quality in the Application of Bladder Cancer Quality of Life Research.

Patient-reported outcomes (PRO), including health-related quality of life (HRQOL) measures, represent important means for evaluating patients' health outcomes and for guiding health care decisions made by patients, practitioners, investigators, and policy makers. In spite of the large number of studies examining HRQOL in patients with bladder cancer, very few review articles investigated this topic. Because these review studies report mixed results, incorporating bladder cancer HRQOL measures into standard urological practice is not a viable option. In this non-systematic review of the literature and commentary we note some general concerns regarding PRO research, but our primary focus is on the HRQOL methodology within the context of two types of bladder cancer: muscle invasive and non-muscle invasive bladder cancer. Considering bladder cancer HRQOL as the interaction of four areas of the assessment process (i.e., what model of HRQOL to choose, what instruments are available to fit the choice, how interpretation of the resulting data fits the model, and how to derive some utility from the chosen model) and the two types of disease (i.e., muscle invasive and non-muscle invasive) may move us toward a better understanding of bladder cancer HRQOL. Establishing a useful model of perceived general health or specific symptoms is the first and most important step in developing the responsive bladder cancer HRQOL measures necessitated by clinical settings.

Effects of thyroid hormone deficiency and replacement on rat hypothalamic growth hormone (GH)-releasing hormone gene expression in vivo are mediated by GH.

The role of thyroid hormone and GH in the regulation of hypothalamic GH-releasing hormone (GRH) gene expression in the rat was examined after the induction of thyroid hormone deficiency by thyroidectomy. Thyroidectomy resulted in a time-dependent decrease in hypothalamic GRH content, which was significant by 2 weeks postoperatively, and a reduction in pituitary GH content to 1% of the control level by 4 weeks. In contrast, GRH secretion by incubated hypothalami under both basal and K(+)-stimulated conditions was increased after thyroidectomy. Hypothalamic GRH mRNA levels also exhibited a time-dependent increase, which was significant at 1 week and maximal by 2 weeks after thyroidectomy. Administration of antirat GH serum to thyroidectomized rats resulted in a further increase in GRH mRNA levels. T4 treatment of thyroidectomized rats for 5 days, which also partially restored pituitary GH content, lowered the elevated GRH mRNA levels. However, comparable effects on GRH mRNA levels were observed by rat GH treatment alone. These results suggest that the changes in hypothalamic GRH gene expression after thyroidectomy in the rat are due to the GH deficiency caused by thyroidectomy, rather than a direct effect of thyroid hormone on the hypothalamus, since the changes were reversible by GH alone despite persistent thyroid hormone deficiency. In addition, they further support the role of GH as a physiological negative feedback regulator of GRH gene expression.

Feedback regulation of growth hormone (GH)-releasing hormone gene expression by GH in rat hypothalamus.

The role of the pituitary, and in particular, of GH in GH-releasing hormone (GRH) gene expression was studied in hypophysectomized rats with and without GH treatment. Hypothalamic GRH mRNA was 6-fold greater in hypophysectomized than in control rats. Increased levels of GRH mRNA were observed at 3 days and the maximal increase was noted at 7 days, postoperatively. Administration of GH to hypox rats partially reversed the increase in GRH mRNA, suggesting a negative feedback regulation by GH of GRH gene activity at the transcript accumulation level. The overall regulation of GRH gene expression, however, appears more complex since GRH mRNA levels and GRH content exhibited discordant changes after both hypophysectomy and GH treatment, suggesting that factors other than GH are required for efficient translation of GRH mRNA.

Tissue-specific transcription initiation and effects of growth hormone (GH) deficiency on the regulation of mouse and rat GH-releasing hormone gene in hypothalamus and placenta.

Hypothalamic GRH gene expression has been shown to be negatively regulated by GH in both rat and mouse. The recent reports of different 5' untranslated sequences in mouse GRH cDNA from hypothalamus and placenta have raised the possibility of tissue-specific regulation of the GRH gene. To provide support for this possibility, we have studied rodent models with GH deficiency due to genetic defects in the pituitary. Complementary DNA probes for the hypothalamic and placental 5' regions were used to determine the tissue specificity of each mRNA. Although the hypothalamic form of GRH mRNA was detected in placenta, it constituted less than 0.7% of total placental GRH mRNA. A placental 5' probe (based on the previously reported sequence) hybridized only with a larger mRNA species and was not tissue specific, indicating that it was not related to GRH and was derived possibly from a cloning artifact. The correct 5' sequence of mouse placental GRH cDNA was determined and shown to be distinct from both that previously reported and the hypothalamic sequence. Although the placental form of GRH mRNA was detected in hypothalamus using the polymerase chain reaction, its levels were undetectable by Northern blotting. The 5' end of rat placental GRH cDNA was similarly sequenced and shown to exhibit no homology with the rat 5' hypothalamic sequence, but a high degree of homology with the corresponding mouse placental sequence. In GH-deficient dwarf (dw/dw) rats, hypothalamic GRH mRNA levels were significantly increased above control levels in both females and males, and pregnancy did not alter the levels in either (dw) or control rats.(ABSTRACT TRUNCATED AT 250 WORDS)

Cloning and characterization of mouse growth hormone-releasing hormone (GRH) complementary DNA: increased GRH messenger RNA levels in the growth hormone-deficient lit/lit mouse.

We have isolated and cloned the full length cDNA for mouse GH-releasing hormone (mGRH) from mouse hypothalamus using a recently described strategy involving the polymerase chain reaction technique (PCR). Degenerate oligonucleotide primers were selected based on short (six amino acids) conserved regions in the human and rat GRH peptides that would recognize DNA sequences encoding similar amino acids regardless of codon usage. Primer-extended cDNA was amplified by PCR on cDNA templates prepared by reverse transcribing total mouse hypothalamic RNA. After cloning and sequencing the initial product, the 3' and 5' ends of mGRH were generated using a separate PCR strategy (RACE protocol). The mGRH cDNA encodes a 103-amino acid reading frame, structurally similar to the human and rat GRH genes, containing a signal sequence, a 42-residue GRH peptide, and a 31-residue C-terminal region. Although the structures of mouse and rat GRH are highly conserved in the signal peptide and C-terminal region, there is considerable diversity in the GRH region, which exhibits nearly comparable homology with the rat (68%) and human (62%) structures. Differences between mouse and rat GRH were also found in the amino acid cleavage sites at the 5' and 3' ends of the mature peptide and at the polyadenylation signal.(ABSTRACT TRUNCATED AT 250 WORDS)

Characteristics of a novel rat anti-mouse platelet monoclonal antibody: application to studies of megakaryocytes.

Using murine platelets as an immunogen, a rat monoclonal antibody (designated 4A5) that recognizes only murine blood platelets and marrow megakaryocytes was developed. The extent of binding of 4A5 to platelets was dependent upon their state of activation. Following phorbol ester, ionophore, or thrombin stimulation of resting platelets, a decrease of > 50% in the binding of 4A5 was observed by flow cytometry. This decrease in antibody binding to the platelets was accompanied by an increase in antibody released into the platelet-free supernatant following platelet activation. When platelets were first radioiodinated, followed by activation and incubation of the platelet-free supernatant with 4A5-derivatized beads, no precipitable counts were observed compared with control resting platelets. This suggests that antibody release was related to an activation-dependent conformational change in the 4A5 epitope. Following solubilization of biotinylated platelets, 4A5 bound to an 80-kd membrane protein. Immunohistochemical studies with 4A5 showed that megakaryocytes could be identified both in vitro and ex vivo. When marrow was first stained histochemically with 4A5 followed by staining for acetylcholinesterase, the distribution of stained cells was similar. Flow cytometric analysis using 4A5 and propidium iodide showed that the antibody could be used to identify megakaryocytes for ploidy analysis in vivo or in vitro. 4A5 was capable of inducing a moderate thrombocytopenia in mice compared with polyclonal anti-platelet serum. When bound to plastic or to magnetic beads, 4A5 could be used to purify murine megakaryocytes to homogeneity. The data suggest that monoclonal antibody 4A5 will be useful in quantitative studies of murine platelets and megakaryocytes.

Canine megakaryocytopoiesis: analysis utilizing a monoclonal antibody to a 140-kd dog platelet protein.

The dog, a convenient and relatively large animal whose size permits repetitive blood and marrow sampling, marrow biopsy, and mechanical apheresis, would be a suitable experimental model for the study of in vivo megakaryocytopoiesis. A monoclonal antibody to a 140-kd dog platelet membrane protein has been developed that reacts with canine megakaryocytes. Using the fluoresceinated derivative of this antibody to identify megakaryocytes and propidium iodide staining to measure relative DNA content, the DNA distribution of megakaryocytes in dog bone marrow or in cultured dog marrow cells could be rapidly assessed by flow cytometry. Normal dogs showed a modal ploidy of 16N (54%), with 17% 8N and 16% 32N. In contrast, dogs made thrombocytopenic by plateletpheresis showed a shift in distribution to higher ploidy cells (36% 32N). The data show that use of a specific marker of megakaryocytes in combination with flow cytometric analysis is an accurate and reproducible method of assessing megakaryocytopoiesis in a convenient and easily manipulable animal model.

Expression of a foreign protein in human megakaryocytes and platelets by retrovirally mediated gene transfer.

Recent progress in the culture of human megakaryocytes (MKs) has led to the capacity to produce platelets in vitro. This capability enables investigation into the possibility of modifying platelet structure and/or function by genetically altering the MK. To this end, a cDNA for the murine CD9 (mCD9) cell surface protein was introduced into MK progenitors by retrovirally mediated gene transfer and subsequently detected in cultured MKs with a monoclonal antibody (MoAb) that specifically recognizes the murine protein. CD34+ human peripheral blood or marrow progenitors, enriched by immunomagnetic bead selection, were cultured for 5 days in the presence of growth factors, including stem cell factor and thrombopoietin, to induce MK progenitors into the cell cycle. The stimulated cells were then cocultured with the mCD9 retroviral producer cell line for 3 days, followed by culture in serum-depleted medium for 3 to 7 additional days. Flow cytometry analysis using the anti-CD9 MoAb and TAB, a MoAb recognizing human GPIIb, revealed that a large proportion (40-100%) of the MKs expressed mCD9. To ascertain whether these cells were capable of producing mCD9+ platelets, flow cytometry analysis was performed at a time when proplatelets were observed in the culture. mCD9 was detected in up to 59% of the TAB+ platelet-sized particles. Because deteriorating MKs can produce platelet-sized particles in vitro, experiments were performed to determine whether mCD9+ TAB+ particles were functionally active. Addition of phorbol myristate acetate resulted in the redistribution of P-selectin (CD62) from the alpha granule to the platelet surface as detected by MoAbs S12 and G5 in three-color flow cytometry analyses. These studies showed that up to 76% of the mCD9+ TAB+ particles were functionally active. The data show that retrovirally mediated gene transfer is a viable approach for genetically altering MK progenitors, resulting in platelets that express heterologous proteins.

Evidence for a defect in growth hormone-releasing factor signal transduction in the dwarf (dw/dw) rat pituitary.

Dwarf (dw/dw) rats exhibit a 40% reduction in body growth, isolated GH deficiency (less than 5% of normal pituitary content), and a decreased number of pituitary somatotrophs (15-20% of normal). Since GH-releasing factor (GRF) stimulates GH synthesis and secretion and somatotroph proliferation, and its effects are probably mediated by cAMP, we have assessed GH secretion and cAMP production in dw rat pituitaries in response to various GH secretagogues. Dispersed pituitary cells from dw rats were less sensitive (2.5-fold) to stimulation of GH secretion by GRF and showed a 25% reduction in the maximal GH response even after normalization of their reduced GH content. Intracellular cAMP was elevated 63-fold over basal levels in normal cells after 4 h in response to maximal GRF stimulation, but only 1.9-fold in dw cells, and even larger differences between the groups were found at earlier time points. The GH responses of dw cells to exogenous cAMP, however, were indistinguishable from normal. Forskolin, a direct stimulator of adenylate cyclase, elicited comparable maximal GH and cAMP responses, but an increased ED50, in dw cells. Activation of GS alpha by cholera toxin showed an increased ED50 and reduced GH and cAMP responses in dw cells, and marked decreases in these responses were observed in response to prostaglandin E1. Phorbol ester stimulation resulted in a reduced maximal GH response in dw cells without a change in sensitivity. These results provide evidence for a defect in the GRF signal transduction pathway associated with a decreased ability of GS alpha to stimulate adenylate cyclase in dw rat somatotrophs that may be causally linked to their GH deficiency.

Growth hormone-releasing hormone immunoreactivity in mouse placenta, maternal blood, and amniotic fluid: molecular characterization and secretion from primary cell cultures in vitro.

The GH-releasing hormone (GRH) gene, along with those of many other hypothalamic hormones, is abundantly expressed in mouse and rat placenta. The presence of GRH immunoreactivity (GRH-IR) is described in mouse placenta, maternal blood, and amniotic fluid, and its molecular form has been characterized using HPLC. Two different molecular forms of mouse GRH-IR (mGRH-IR) were detected in the mouse hypothalamus and one in placenta. Twenty-five percent of mGRH-IR in the hypothalamus corresponded to mGRH(1-42)OH, whereas the remainder, and all of the mGRH-IR in placenta, had a retention time consistent with the GRH precursor. High levels of mGRH-IR were detected in both maternal plasma and amniotic fluid. In addition, a mouse placental cell primary culture system was established to study the regulation of mGRH-IR release. Turnover of mGRH in placental cells was rapid, resulting in a 24-h media content of 10 times that present in cells. Both 1-oleoyl-2-acetyl-sn-glycerol and 1,2-dioctanoyl-sn-glycerol significantly stimulated the release of mGRH-IR from cultured placental cells into the incubation media but had no effect on total peptide synthesis. These results suggest that the release of mGRH-IR from placental cells is mediated, at least in part, by the activation of protein kinase C. The HPLC elution profiles of mGRH-IR released from placental cells under basal and 1-oleoyl-2-acetyl-sn-glycerol-stimulated conditions were similar to those in placental tissue. Although the biological function of mGRH-IR in placental, maternal plasma, and amniotic fluid is not yet clear, the presence of mGRH-IR in these tissues and circulating fluids suggests the possibility that mGRH-IR may exert an important role in both fetal and maternal physiology.

Inhibitory effect of hypothalamic medial preoptic area somatostatin on growth hormone-releasing factor in the rat.

Possible inhibitory effects of somatostatin (SRIF) on GRF were studied by assessing spontaneous GH secretion and GRF content and release in adult male rats depleted of hypothalamic SRIF by anterolateral hypothalamic deafferentation (AHD) or electrolytic lesions in the medial preoptic area (MPO). Plasma GH levels were measured 7 days postoperatively every 20 min in conscious animals with indwelling iv cannulae. Median eminence SRIF was markedly reduced 8 days postoperatively in both AHD and MPO rats, as determined by immunohistochemistry and RIA (P less than 0.01). Although GRF immunoreactivity in the median eminence of AHD and MPO animals appeared well preserved immunocytochemically, hypothalamic GRF content by RIA was significantly decreased at 8 days (P less than 0.01). Spontaneous GH secretion was pulsatile in sham-operated animals. In contrast, basal GH levels in AHD and MPO animals were markedly elevated (P less than 0.01), and secretory pulses were absent. Intravenous injection of specific anti-GRF serum into MPO animals decreased the elevated plasma GH levels (P less than 0.01), indicating increased hypothalamic GRF secretion. GRF release from hypothalamic median eminence-arcuate nucleus complexes in vitro was significantly greater in AHD and MPO animals than in control animals 4 and 8 days postoperatively in response to 30 mM K+ (P less than 0.01), but not under basal conditions. These results suggest that hypothalamic medial preoptic area somatostatinergic neurons play a tonic inhibitory role in the regulation of GRF release and that GH hypersecretion observed after MPO and AHD is attributable to changes in both SRIF and GRF.