Evidence for the existence of substance P in the prepubertal rat ovary. II. Immunocytochemical localization

Nerve fibers containing substance P (SP) were localized in ovaries from juvenile and peripubertal rats by immunofluorescence. These fibers were closely associated with the theca externa of antral follicles, as well as being in the interstitial tissue and within the tunica adventitia of small blood vessels, mostly arterioles. Consistently, the greatest amount of SP immunoreactivity was observed surrounding the ovarian vasculature. Substance P was not detected in cells or within the corpora lutea (CL). Additionally, the peripubertal animals seemed to have a greater concentration of ovarian SP than the juvenile animals. Possible functional roles for this peptide in the ovary are discussed.     

Evidence for the existence of gonadotropin receptors in the nuclei isolated from rat ovary

Specific binding of radiolabeled human chorionic gonadotropin (hCG) to nuclei isolated from pseudopregnant rat ovaries was studied. Incubation of cultured luteal cells or isolated nuclei with fluorescein isothiocyanate conjugated hCG showed concentration of fluorescence in the nuclear region. Isolated nuclei exhibited saturable high affinity binding of radiolabeled hCG with an apparent Kd of 3.42 X 10(-10) M. The binding was inhibited by increasing concentrations of unlabeled hCG. Under dissociating conditions, the bound hCG was dissociated from the nuclei. However, unlike the plasma membranes, the hCG bound to nuclei was not degraded before dissociation. Radiolabeled hCG bound to the nuclei could also be dissociated by brief exposure to MgCl2 or acidic incubation medium. The bound hCG was not extractable with 4M KCl or 2% Triton X-100. The available evidence suggest that nuclear receptors are distinct from plasma membrane receptors for hCG.     

Sequence analysis of cloned cDNA for rat substance P precursor: existence of a third substance P precursor

The sequence of the mRNA for the rat substance P precursor (preprotachykinin A) has been elucidated by molecular cloning and sequence analysis. The deduced amino acid sequence of rat preprotachykinin A indicates that it contains both substance P and substance K but differs in the sequence organization from either bovine alpha- or beta-preprotachykinin A reported previously. The existence of the bovine mRNA for the third preprotachykinin A has thus been examined and evidenced by the isolation of the corresponding cDNA clone. This mRNA, named gamma-preprotachykinin A mRNA, deletes the sequence precisely corresponding to the exon 4 sequence of the preprotachykinin A gene. Thus, alternative RNA splicing in the expression of the single preprotachykinin A gene results in the generation of three different forms of the preprotachykinin A mRNAs.     

Anti-implantation role for substance P and neurotensin in rat.

Substance P (SP) and neurotensin (NT), two structurally related peptides with contrasting biological actions, have been shown to have some role in peripheral reproductive processes. Intrauterine microinjection of SP or NT on day 4 or 5 of pregnancy in the rat significantly reduced the number of viable fetuses, weight and glycogen content of the uterus. The number of viable fetuses, uterine weight or glycogen content were not modified when SP/NT was microinjected on day 8, 9, 10 or on day 14, 15 and 16. The results indicate that the peptides possibly exert a direct local alteration in uterine vascular permeability causing failure in implantation.     

Renal inactivation of substance P in the rat.

The activity and distribution of substance P-catabolizing enzyme(s) were studied in the rat kidney. Kidney homogenates inactive substance P 5-20 times as fast as do homogenates of intestine, liver, lung, heart or brain. The catabolizing activity was highest in the cortex and decreased progressively down the papilla. Cortex of rat kidney was homogenized and fractions enriched in microsomal membrane, final supernatant, plasma membrane, endoplasmic reticulum, brush border and intact glomeruli were prepared. The identity and homogeneity of the preparations were determined by assaying marker enzymes and by morphological examination. Substance P was catabolized most rapidly by the microsomal and plasma-membrane-enriched fractions, and least rapidly by endoplasmic reticulum or final supernatant fractions. Purified brush border of proximal tubules inactivated substance P more than 10 times as fast as isolated glomeruli. Our experiments show that substance P is catabolized at a rate that is similar to the rates of inactivation of bradykinin and angiotensin II. Further, the distribution of substance P-catabolizing activity in various kidney fractions is similar to the distribution of kininase and angiotensinase activities previously reported.     

Biological activity and enzymic degradation of substance P analogs: implications for studies of the substance P receptor.

Partial sequences of Substance P, either free or blocked at their amino terminal, have been examined for their stability towards inactivation by homogenate or particulate fractions of rat brain and for their relative potencies as smooth muscle contractors. The C-terminal hexapeptide in both the free and blocked forms displays activity comparable to that of the longer C-terminal peptides as well as to that of the native undecapeptide. The blocked peptides, however, are much more stable than their corresponding free peptides. Among the free peptides Substance P is degraded slower than the free hexa- and hepta-peptides, suggesting that the N-terminal tetrapeptide part may play a role in stabilizing the molecule. Blocked hepta- and octapeptide analogs, carrying probe properties, may be useful for studies of the Substance P receptor.     

Biochemical studies of rat liver Golgi apparatus. I. Isolation and preliminary characterization.

A method is described for the isolation of morphologically well-preserved Golgi apparatus from rat liver. The method is essentially the same as that of Morré et al. (Morré, D.J., Hamilton, R.L., Mollenhauser, H.H., Mahley, R.W., Cunningham, W.P., Cheetham, R.D., & Lequire, V.S. (1970) J. Cell Biol. 44, 484-491) except that mild cell disruption is achieved by means of a stainless-steel sieve. The average recoveries of protein and galactosyltransferase in the isolated fraction are about 6 mg from 10 g of perfused liver and about 35% from the homogenate, respectively. The preparation is virtually free from succinate-cytochrome c reductase, glucose-6-phosphatase, acid phosphatase, and 5'-nucleotidase. The Golgi fraction as well as its vesicular fragments is homogeneous upon isopycnic centrifugation in both sucrose and dextran density gradients. Their buoyant densities in sucrose are significantly higher than those in dextran, indicating that both forms of the organelle are closed systems which are impermeable to macromolecules. The galactosyltransferase activity of a freshly prepared Golgi fraction, measured with ovalbumin as galactosyl acceptor, is activated 26-fold by the addition of Triton X-100, whereas those of homogenized, sonicated, and aged preparations are only activated 2- to 4-fold.     

Expression and localization of P-, K-, and OB-cadherin in the prepubertal rat ovary.

Classical and atypical cadherins mediate calcium-dependent cell adhesion and play an important role in morphogenetic processes. We have shown, previously, N- and E-cadherin expression in the rat ovary. This expression, however, was not associated with specific follicle-restructuring events such as antrum formation and segregation of mural from cumulus granulosa cells suggesting that other cadherins may serve this function. In this study, RT-PCR and immunostaining techniques showed that three other cadherins are expressed throughout prepubertal ovarian development in the rat: one classical (P-) cadherin, and two atypical (K- and OB-) cadherins. RT-PCR analysis of isolated ovarian tissue compartments (granulosa cells and the residual ovarian tissue) agreed with the immunostaining results. Immunostaining showed P- and K-cadherin expression by granulosa, as well as thecal/interstitial cells, and also in oocytes of primordial follicles. P-cadherin expression was absent in oocytes of follicles in later stages of development compared to K-cadherin, which was found in oocytes at all stages of folliculogenesis. P-, K-, and OB-cadherin were expressed by the ovarian surface epithelial cells of neonatal animals but only P- and OB-cadherin expression were maintained in these cells in 25 day-old animals. Cellular OB-cadherin staining was absent in follicles at all stages of development and its expression was restricted to the ovarian hilar region and portions of the stroma. In summary, cadherin expression and distribution profiles changed during ovarian growth and folliculogenesis suggesting a role for cadherins in organizational and morphogenetic processes within the developing rat ovary.     

Biosynthesis of serum albumin in rat liver. Evidence for the existence of `proalbumin''

1. A protein(s) of rat liver (precipitated from soluble extracts of the microsomal fraction by anti-albumin) yields albumin after limited hydrolysis by trypsin. 2. Evidence that the product of limited tryptic hydrolysis is albumin, is based upon ion-exchange chromatography, electrofocusing and peptide `mapping'. 3. The albumin `precursor' is recognized by anti-albumin and is apparently not distinguished from albumin by anti-albumin. 4. A small peptide is liberated from the presumptive albumin precursor during limited tryptic hydrolysis. This peptide is labelled by arginine, but not by leucine, lysine or methionine. 5. These results support our previous suggestion based on kinetic evidence that the albumin-like protein(s), in the anti-albumin precipitate from rat liver, is an albumin precursor.     

Binding studies of substance P anterior pituitary binding sites: changes in substance P binding sites during the rat estrous cycle

Previous studies have shown that substance P (SP), an undecapeptide widely distributed in the gastrointestinal tract and in the peripheral and central nervous system, is a putative regulatory peptide involved in the control of reproductive function. Specifically, SP inhibited, at the anterior pituitary (AP) level, the stimulatory action of a physiological concentration (10(-8) M) of Gonadotropin Releasing Hormone (GnRH) on the release of the luteinizing hormone (LH). In the present work, we have demonstrated the presence of specific SP binding sites in the AP and related changes in the number of these sites to GnRH receptor number, hypothalamic SP and GnRH content and LH secretion during the rat estrous cycle. High affinity saturable SP binding sites (Kd, 1.5 approximately equal to 10 nM) were demonstrated in AP membranes using [3H]-SP or a novel analog, [125I]-(D-Tyr0, NorLeu11)SP. The binding affinity of SP fragments decreased with progressive removal of amino acid residues from N or C termini of the molecule. Other neuropeptides had low affinity for the SP binding sites. During the rat estrous cycle, SP and GnRH binding capacity of the anterior pituitary were inversely related. At the time of the proestrous LH surge, the AP binding capacity was low for GnRH but high for SP. The highest content of SP in the hypothalamus were recorded during the afternoon of proestrus when hypothalamic GnRH levels were lowest and the preovulatory surge occurred. These studies have established the presence of high affinity specific binding sites for SP in the AP which alter during the estrous cycle in a manner appropriate for mediating the direct inhibitory effects of SP on LH release in vitro.     

Evidence for the existence of actomyosin ATPase in the rat pancreas. Isolation and biochemical characterization

In a crude extract of rat pancreas, myosin was associated with a protein having the same electrophoretic mobility as actin. This myosin was purified after dissociation of the actomyosin complex with KI-ATP. On sodium dodecylsulfate/acrylamide gel electrophoresis, the isolated pancreatic myosin showed a major component of approximately 200 kDa, and two smaller components with apparent molecular weight of 22 and 15 kDa, respectively. This purified myosin exhibited high ATPase activity in the presence of K+ + EDTA or Ca2+ and very little activity in the presence of Mg2+. (K+ + EDTA)-ATPase activity showed one pH optimum at 8.0, while Ca2+-ATPase activity showed two pH optima at 6.0 and 9.0, respectively. (K+ + EDTA)-stimulated enzyme activity was specific for ATP whereas Ca2+-stimulated activity showed low specificity for nucleoside triphosphates.     

Evidence for the existence of multipotential lympho-hematopoietic stem cells in adult rat

Rats were given near-lethal doses of x-ray to produce clones of hemic cells marked by radiation-induced chromosome abnormalities. Subsequently, bone marrow from these rats was injected into lethally irradiated mice to form erythropoietic spleen colonies; and peripheral blood lymphocytes from the same rats were stimulated to proliferate in a mixed lymphocyte interaction (MLI), an immunological response to histocompatibility isoantigens. Chromosome markers indicated that in several instances the cells of an erythroid spleen colony and a proportion of the lymphocytes reacting in the MLI were progeny of the same stem cell in the donor rat. In addition, lymphocytes of the same radiation-marked clone were shown to proliferate in response to several different histocompatibility isoantigens. The data indicate the presence in the adult rat of a primitive lymphohematopoietic stem cell capable of yielding both erythroid and lymphoid progeny. The findings also suggest that immunological specificity is determined during lymphoid differentiation, subsequent to the stem cell stage.     

Electron microscopic evidence for the existence of an intercellular substance in rat cerebral cortex

Summary The intercellular spaces of rat cerebral cortex are filled with a dense material, demonstrable by electron microscopy. This intercellular substance is in part preserved by chemical fixation with formaldehyde and osmium tetroxide but is solubilized and largely lost during subsequent dehydration with ethyl alcohol. Dehydration with acetone or Durcupan favors the preservation of the intercellular substance, which is preserved also by freezing and drying. Whether the intercellular substance demonstrated here is part of the outer leaflets of apposing plasma membranes (glycocalyx) or truly an intercellular substance similar to connective tissue ground substance is not known. The probability of the latter is discussed with regard to proposed physiological mechanisms.This work was supported by USPHS Research Grants NB 05175 and AM 06998.     

Effects of capsaicin on nitric oxide synthase isoforms in prepubertal rat ovary

Nitric oxide (NO) has emerged as an important intra-ovarian regulatory factor. We investigated effects of low dose capsaicin (CAP) treatment on the different NOS isoforms in prepubertal rat ovaries. Fifteen 21-day-old female Sprague-Dawley rats were divided randomly into three groups. The first group received no treatment, the second group received 0.5 mg/kg/day CAP dissolved in the vehicle, and the third group was treated with the vehicle only. The animals were euthanized by ether inhalation after 15 days and their ovaries were excised. Ovaries were fixed in 10% neutral buffered formalin and embedded in paraffin. Sections were processed for standard immunohistochemistry using the labeled streptavidin-biotin technique for expression of nNOS, eNOS and iNOS. We demonstrated that CAP induced expression of NOS isotypes including eNOS, iNOS and nNOS in prepubertal rat ovaries. CAP may lead to release of NO either directly from nerves or indirectly by evoking release from other cells via the action of neuropeptides that are released from afferent terminals and are involved in regulating female reproductive function.     

Multiple transmitter neurons in Tritonia. I. Biochemical studies

The buccal ganglia of the marine mollusc Tritonia control a variety of movements associated with feeding, including gut motility. The buccal ganglia and gut contain a class of peptides termed small cardioactive peptides (SCPs). Cobalt backfilling of the nerve which innervates the gut stains several buccal neurons including two pairs of reidentifiable cells, B11 and B12. Both appear white under epiillumination, a characteristic of peptidergic neurons in gastropods. Enzymatic and biochemical analyses of extracts from microdissected B11 cell bodies demonstrate that this neuron contains two species of SCPs. Labeling in organ culture followed by dissection and extraction of cell bodies indicates that these peptides were synthesized in B11. One of these peptides appears to be identical to SCPB, one of two SCPs that have been sequenced. The other SCP present in these neurons is novel. Less extensive analyses of extracts of B12 somata suggest that it also contains the same SCPs. In addition to the peptides, B11 also contains large quantities of acetylcholine (ACh) as determined by a radioenzymatic assay of cell body extracts. B12 does not contain measureable ACh. The concentration of the two peptides and ACh in the B11 cytoplasm is approximately 1 mM. Neuron B11 appears to be an appropriate model system for studying the biochemical and physiological properties of multiple transmitter neurons.     

Effects of capsaicin on nitric oxide synthase isoforms in prepubertal rat ovary

Abstract

Nitric oxide (NO) has emerged as an important intra-ovarian regulatory factor. We investigated effects of low dose capsaicin (CAP) treatment on the different NOS isoforms in prepubertal rat ovaries. Fifteen 21-day-old female Sprague-Dawley rats were divided randomly into three groups. The first group received no treatment, the second group received 0.5 mg/kg/day CAP dissolved in the vehicle, and the third group was treated with the vehicle only. The animals were euthanized by ether inhalation after 15 days and their ovaries were excised. Ovaries were fixed in 10% neutral buffered formalin and embedded in paraffin. Sections were processed for standard immunohistochemistry using the labeled streptavidin-biotin technique for expression of nNOS, eNOS and iNOS. We demonstrated that CAP induced expression of NOS isotypes including eNOS, iNOS and nNOS in prepubertal rat ovaries. CAP may lead to release of NO either directly from nerves or indirectly by evoking release from other cells via the action of neuropeptides that are released from afferent terminals and are involved in regulating female reproductive function.