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Orexins are recently discovered neuropeptides that play an important role in the regulation of hormone secretion, and their receptors have been recently demonstrated in the pituitary. The effects of orexin-A on voltage-gated Ca2+ currents and GH release in primary cultured ovine somatotropes were examined. The expression of orexin-1 receptor was demonstrated by RT-PCR in ovine somatotropes, from which Ca2+ currents were also isolated as L, T, and N currents. Application of orexin-A (100 nM) significantly and reversibly increased only the L current, and coadministration of orexin-A and GHRH (10 nM) showed an additive effect on this current, but no effect of orexin-A was observed on either T or N current. Furthermore, the orexin-A-induced increase in the L current was completely abolished by the inhibition of protein kinase C (PKC) activity using calphostin C (100 nM), phorbal 12,13-dibutyrate pretreatment (0.5 micro M) for 16 h or specific PKC inhibitory peptide PKC(19-36) (1 mM). However, the increase in L current by orexin-A was sustained when cells were preincubated with a specific protein kinase A blocker H89 (1 micro M) or a specific intracellular Ca2+ store depleting reagent thapsigargin (1 micro M). Finally, orexin-A alone did not significantly increase GH release, but coadministration of orexin-A and GHRH showed a synergistic effect on GH secretion in vitro. Our results therefore suggest that orexin-A may play an important role in regulating GHRH-stimulated GH secretion through the enhancement of the L-type Ca2+ current and the PKC-mediated signaling pathway in ovine somatotropes.

Intracellular thyroid hormone concentration and action were examined in the liver and the pituitary of a nephrectomized rat model (Nx); the results were compared with those obtained from control (C), thyroidectomized (Tx), and nephrectomized-thyroidectomized (NxTx) littermates. Based on the severity of the uremia, Nx rats were subdivided into Nx1 and Nx2 groups; the former included rats with serum urea nitrogen of less than 100 mg/dl and the latter rats with serum urea nitrogen greater than 100 mg/dl. A group of rats pair-fed to the Nx rats was also included (PF). In the liver, nuclear T3 content (picograms per g liver) and T3-receptor binding capacity (Cmax, picograms T3 per mg DNA) were measured. The respective results from all groups of rats were as follows (asterisks denote values differing from C with a P value less than 0.05): C, 308 +/- (SE) 45 and 121 +/- 11; Nx1, 245 +/- 43 and 85 +/- 15; Nx2, 163 +/- 19 and 74 +/- 7; Tx, 43 +/- 11 and 91 +/- 10; NxTx, 33 +/- 10 and 54 +/- 6; and PF, 237 +/- 20 and 121 +/- 9. T3 receptor binding affinity (Ka), ranging from 3.62-5.28 X 10(9) M-1, was not significantly different among the six groups of rats. In the pituitary, T3 content (picograms per mg pituitary) was reduced only in the Tx rats, being 3.13 +/- 0.89 as compared to 7.04 +/- 1.48 in the C rats (P less than 0.05). In the Nx1, Nx2, and PF rats, pituitary T3 contents were 9.81 +/- 3.22, 13.01 +/- 3.60, and 7.83 +/- 1.08, respectively, and were not different from the C rats. Serum TSH was reciprocally elevated only in the Tx rats. The reduction in hepatic nuclear T3 content and T3-Cmax in the Nx2 rats is consistent with the presence of selective tissue deficiency of thyroid hormone. This is in agreement with the observation of reduced activity of two liver enzymes known to be under thyroid hormone regulation. The pituitary, however, had normal T3 content, suggesting a dissociation in thyroid hormone-dependent metabolic status between a peripheral tissue (liver) and the pituitary. This explains the failure to observe an increase in the serum TSH level, a manifestation of reduced intracellular rather than serum T3 concentration. Decreased food intake appeared not to be the cause of thyroid hormone abnormalities observed in uremia, as PF rats failed to manifest the changes found in Nx rats.

Surfactant protein A (SP-A), with a reduced denatured molecular mass of 26-38 kDa, is characterized by a collagen-like sequence in the N-terminal half of the protein. This protein forms an oligomeric structure which is dependent upon this collagenous domain. SP-A has been demonstrated to function as an inhibitor of phospholipid secretion by primary cultures of alveolar type II cells via a cell surface receptor for the protein. However, the receptor-binding domain of SP-A has not been identified. The purpose of the present study was to investigate the role of the C-terminal domain of SP-A in binding to type II cells and regulation of phospholipid secretion. A monoclonal antibody to human SP-A, whose epitope was localized at the C-terminal domain of the protein, abolished the inhibitory activity of human SP-A on lipid secretion by type II cells, and attenuated the ability of human SP-A to compete with 125I-(rat SP-A) for receptor binding. SP-A was then digested with collagenase and the collagenase-resistant fragment (CRF), which is the C-terminal domain of SP-A (thus lacking the N-terminal domain), was isolated. Gel filtration chromatography revealed that CRF exists as a monomer in solution containing Ca2+. CRF had the ability to inhibit phospholipid secretion, although at a higher concentration than for SP-A, and was also able to compete with 125I-(rat SP-A) for binding to type II cells. A direct binding study showed that CRF bound to type II cells in a concentration-dependent manner. The present study demonstrates that the non-collagenous, C-terminal, domain of SP-A is responsible for the protein's inhibitory effect on lipid secretion and its binding to type II cells.

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