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Biomedical subjects

C R Hopkins

Publications and source records attributed to C R Hopkins.

At least 73 records · Page 4Linked to original sources
PubMed1979

Cellular origins of different forms of gastrin. The specific immunocytochemical localization of related peptides.

We have localized the antigenic determinants for the main forms of gastrin (big gastrin, G34, and little gastrin, G17) in hog antral mucosa using sequence specific antibodies and an indirect immunofluorescence technique. Populations of monospecific antibodies were obtained after affinity immunoadsorption to remove populations of unwanted specificity. The specificity of the purified antisera was established by direct binding of 125I labeled peptides to antisera at the same dilutions as those used in immunocytochemistry. The results indicate that in hog antral mucosa there is a single population of cells with the antigenic determinants of the C-terminal region of G17 and G34, the N-terminal region of G17, the N-terminal region of G34, and the intact G17 molecule. In duodenum there are cells with only C-terminal reactivity; since gastrin and CCK share a common C-terminal sequence it is concluded that this cell type contains CCK-like peptides rather than gastrin.

Animals↗
PubMed1979

The identification of urogastrone in serum, saliva, and gastric juice.

Urogastrone, a peptide isolated from human urine, is known to cause inhibition of gastric acid secretion and proliferation of fibroblasts in culture; furthermore immunofluorescent localization techniques show it to be present in submandibular and Brunner's glands. Serum, saliva, and gastric juice samples have now been fractionated upon Sephadex G-200 and G-50 and the immunoreactive urogastrone located using a specific radioimmunoassay. Biologic activity was shown by mitogenic studies with 3T6 fibroblasts. In serum, the major immunoreactive component was ca. 1-2 X 10(5) daltons, but trypsin treatment then gave a smaller biologically active species in the same position as pure urogastrone on Sephadex G-50. Both saliva and gastric juice showed major components at the position defined by urogastrone, and these also stimulated the uptake of [H]thymidine into the fibroblasts. It is concluded that a urogastrone-like molecule can be released enzymically from a high molecular weight serum precursor and that the small biologically active peptide is also a normal component of saliva and gastric juice.

Autoradiography↗
PubMed1978

Discrimination of LH, FSH, TSH and ACTH in dissociated porcine anterior pituitary cells by light and electron microscope immunocytochemistry.

Using the PAP unlabelled antibody method, LH, FSH, TSH and ACTH were localized at light microscope level in cultured cells dissociated from the porcine adenohypophysis. Antisera were shown to be specific for the beta-subunits of the porcine glycoprotein hormones by radioimmunoassay and absorption studies. Using these antisera, it was found that LH and FSH were contained within the same cell, with TSH in a separate cell. When absorbed with LH, anti-porcine ACTH stained a separate distinct population of ACTH cells. Adjacent ultra-thin sections stained with anti-pLH beta and anti-pFSH beta, and examined at electron microscope level, showed that the ovoid, 150--400nm secretory granules of the LH/FSH gonadotrophs contained both LH and FSH.

Adrenocorticotropic Hormone↗
PubMed1978

Dissociation of the porcine anterior pituitary: the kinetics of luteinizing hormone release in response to luteinizing hormone-releasing hormone.

A method has been developed for disaggregating porcine anterior pituitary tissue and for providing dissociated preparations which have good, stable viability in culture. The secretory capacity for these preparations in terms of their ability to release luteinizing hormone in response to luteinizing hormone-releasing hormone has been documented in detail. In following the short-term kinetics of LH secretion a biphasic pattern of release has been demonstrated in which a sharp initial peak, maximal at 2 min, is followed by a second, more prolonged phase of release reaching a maximum between 10 and 20 min.

Animals↗
PubMed1978

Calcium as a second messenger in the stimulation of luteinizing hormone secretion.

In cells dissociated from porcine anterior pituitary glands and maintained in culture for 48 h the specific secretagogue luteinizing hormone-releasing hormone (LH-RH) induces a biphasic pattern of luteinizing hormone (LH) release. A biphasic pattern of release is also induced by 57 X 10(-3) M K+ and the ionophore A-23187. By reducing the availability of Ca2+, either by omission from the medium, chelation or interfering with Ca2+ transport across the plasma membrane, it is shown that LH release stimulated by LH-RH is much less dependent upon the availability of extracellular Ca2+ than that stimulated by either high K+ or A-23187. Nevertheless, by using a lanthanum displacement protocol to follow the influx of 45Ca2+ it is shown that LH-RH stimulation does induce an influx of extracellular Ca2+. Parallel experiments in which the stimulated 45Ca2+ efflux from preloaded cells is followed confirm the influx data but suggest, in addition, that when the influx of extracellular Ca2+ is inhibited, the peptide is able to mobilize Ca2+ from an intracellular location. It is thus concluded that while LH release can be initiated by an increase in the intracellular level of Ca2+, and although LH-RH stimulation does increase the permeability of the plasma membrane to Ca2+, the stimulation of LH release by LH-RH is not dependent upon extracellular Ca2+.

Animals↗
PubMed1977

Short term kinetics of luteinizing hormone secretion studied in dissociated pituitary cells attached to manipulable substrates.

With the use of poly-L-lysine, a method has been developed which induces acutely dissociated rat anterior pituitary cells to attach to glass and polyacrylamide surfaces. In these attached cells the recovery of the secretory response, which is impaired in acutely dissociated cells, has been followed, and it has been established that, in terms of their ability to secrete luteinizing hormone (LH) in response to the specific secretogogue luteinizing-hormone-releasing hormone (LHRH), the cells become maximally responsive after 48 h. The attached cells also allow the short-term kinetics of LH secretion to be followed with great facility; and, when cells allowed to recover for 48 h are used, it is shown that in response to LHRH the pattern of LH release is biphasic.

Animals↗
PubMed1977

Topographical localization of the receptors for luteinizing hormone-releasing hormone on the surface of dissociated pituitary cells.

A derivative of the hypothalamic peptide luteinizing hormone-releasing hormone (LHRH) has been coupled to ferritin and the conjugate purified by gel chromatography. In its ability to stimulate the secretion of luteinizing hormone from pituitary cells in vitro, the conjugate has the same potency and specificity as the native peptide. When dissociated pituitary cells maintained in short-term culture are lightly fixed with formaldehyde and then incubated with the conjugate, examination in the electron microscope shows an even distribution of ferritin particles over the free cell surface of the gonadotrophin cells. This binding appears to be specific for the LHRH receptor since it is prevented by a 10-fold excess of native peptide. In addition to the gonadotrophin cells, some somatotrophin and thyrotrophin cells bind conjugate on their free surfaces under similar conditions. If living cells are incubated with the conjugate for 15 min, the bound conjugate becomes aggregated and then concentrated in one localized area of the cell surface. In this area, which lies immediately above the juxtanuclear Golgi complex, the plasma membrane is frequently invaginated in a manner which suggests that the bound, aggregated conjugate is internalized by endocytosis.

Animals↗
PubMed1975

Caerulein secretion by dermal glands in Xenopus laevis.

Since gastrin and its related peptides are secreted by a minority population of widely dispersed cells in mamamalian tissues it has, in the past, been difficult to study the subcellular aspects of their secretion. From published reports (1, 2) it seemed possible that a satisfactory system for such studies might be provided by the skin of certain amphibians such as Xenopus laevis since in these tissues high concentrations of peptides such as caerulein exist, and there is some indication (3) that this, or a similar gastrin-like peptide, may be a dermal gland secretory product. We have therefore explored this possibility by studying the structure, secretory process, and secretory product of the most prominent non mucous type of gland in the skin of X. laevis. These studies clearly demonstrate that most, if not all, of the caerulein in which the skin is contained in secretion granules within the dermal glands and that its release can be specifically evoked by adrenergic stimulation. The release process by a holocrine mechanism expels all of the stored secretion onto the skin surface and thus for biosynthetic studies it should now be possible to synchronize the processes which lead to the replenishment of the peptide.

Animals↗
PubMed1975

Synthesis and secretion of a large glycoprotein in the pars intermedia.

The biosynthesis, intracellular transport and release of [3-H]-leucine-containing secretory product has been followed in the pars intermedia of Xenopus laevis and, in particular, the synthesis and secretion of a large molecular weight glycoprotein secretory product was demonstrated. However, if Xenopus adrenocorticotrophin does contain a leucine residue the results obtained provided no support for the view that it serves as a precursor for melanocytestimulating hormone in this species.

Adrenocorticotropic Hormone↗
PubMed1973

Hormone secretion by cells dissociated from rat anterior pituitaries.

A new procedure has been developed for dissociating anterior pituitary tissue and producing a viable suspension of single cells. The procedure involves incubation of small tissue blocks in 1 mg/ml trypsin (15 min), followed by incubation in 8 microg/ml neuraminidase and 1 mM EDTA (15 min), followed by mechanical dispersion. Cell yields are approximately 55%, based on recovered DNA. By electron microscopy five types of secretory cells (somatotrophs, mammotrophs, thyrotrophs, gonadotrophs, and corticotrophs) plus endothelial and follicular cells can be identified and are morphologically well preserved up to 20 h after dissociation. Throughout this period, the cells incorporate linearly [(3)H]leucine into protein for up to 4 h at a rate 90% greater than hemipituitaries, and they synthesize, transport intracellularly, and release the two major pituitary secretory products, growth hormone and prolactin. Immediately after dissociation the cells' ability to respond to secretogogues (high K(+) and dibutyryl cyclic AMP) is impaired, but after a 6-12-h culture period, the cells apparently recover and discharge 24% and 52%, respectively, of their content of prelabeled growth hormone over a 3-h period in response to these two secretogogues. This represents a stimulation of 109% and 470% over that released by cells incubated in control medium. The results demonstrate that function and morphologic integrity are preserved in this cell system. Therefore it is suitable for the study of various aspects of pituitary secretion and its control.

Animals↗
PubMed1972

The biosynthesis, intracellular transport, and packaging of melanocyte-stimulating peptides in the amphibian pars intermedia.

Experiments in which glycine-(3)H has been introduced into excised neurointermediate lobes of Xenopus laevis incubated in a modified Krebs-Ringer bicarbonate medium have shown that approximately 50% of the incorporated radioactivity is present in small peptides which have an electrophoretic mobility characteristic of the melanocyte-stimulating (MSH) peptides shown to be elaborated within the tissue. Based on these results and the demonstration that a discrete approximately 7 min pulse of the label can be introduced into the tissue, electron microscope radioautography has been employed to follow the subcellular events concerned with the synthesis, intracellular transport, and packaging of the labeled secretory product. Together, these studies indicate that the newly synthesized material arises in peptide form, rather than as part of a larger prohormone molecule, on the ribosomes of the rough endoplasmic reticulum within the parenchymal cells of the intermediate portion of the lobe. A proportion is then incorporated into and remains for an extended period within the intracisternal granules which are a feature of the rough endoplasmic reticulum within these cells in vitro Most ( approximately 60%) of the labeled secretory product, however, is transferred to the Golgi complex within 30 min and, within a further 10 min, becomes packaged into small ( approximately 200 mmicro) electron-opaque secretory granules. It is probable that under the conditions employed these granules represent the final intracellular location of secretory product before it is released

Animals↗