Ronal R. Macgregor

Effects of Oestrogen on Trigeminal Ganglia in Culture: Implications for Hormonal Effects on Migraine

Although migraine is more common in women than men and often linked to the menstrual cycle, few studies have investigated the biological basis of hormonal influences on the trigeminovascular system. In the present study we investigated the effect of physiological levels (10-9 M) oestrogen on female rat trigeminal ganglia in vitro. Immunocytochemical analysis demonstrated the presence of oestrogen receptor-alpha in a predominantly cytoplasmic location and in neurites. Microarray analysis demonstrated that oestrogen treatment regulates several genes with potential relevance to menstrual migraine. The genes that were upregulated included synapsin-2, endothelin receptor type B, activity and neurotransmitter-induced early gene 7 (ania-7), phosphoserine aminotransferase, MHC-1b, and ERK-1. Down-regulated genes included IL-R1, bradykinin B2 receptor, N- tropomodulin, CCL20, GABA transporter protein, fetal intestinal lactase-phlorizin hydrolase, carcinoembryonic antigen-related protein, zinc finger protein 36, epsin 1 and cysteine string protein. Protein activity assays demonstrated that exposure of the cultured neurons to oestrogen leads to activation of ERK, which has been linked to inflammatory pain. Immunocytochemistry demonstrated that activated ERK was present in neurons containing peripherin, a marker of nociceptive neurons. Several of the genes in the present study may provide potential targets for understanding the association of oestrogen with migraine and other hormone- related orofacial pain.

Adhesion of Pancreatic Beta Cells to Biopolymer Films

Dramatic reversal of Type 1 diabetes in patients receiving pancreatic islet transplants continues to prompt vigorous research concerning the basic mechanisms underlying patient turnaround. At the most fundamental level, transplanted islets must maintain viability and function in vitro and in vivo and should be protected from host immune rejection. Our previous reports showed enhancement of islet viability and insulin secretion per tissue mass for small islets (<125 μm) as compared with large islets (>125 μm), thus, demonstrating the effect of enhancing the mass transport of islets (i.e. increasing tissue surface area to volume ratio). Here, we report the facile dispersion of rat islets into individual cells that are layered onto the surface of a biopolymer film towards the ultimate goal of improving mass transport in islet tissue. The tightly packed structure of intact islets was disrupted by incubating in calcium‐free media resulting in fragmented islets, which were further dispersed into individual or small groups of cells by using a low concentration of papain. The dispersed cells were screened for adhesion to a range of biopolymers and the nature of cell adhesion was characterized for selected groups by quantifying adherent cells, measuring the surface area coverage of the cells, and immunolabeling cells for adhesion proteins interacting with selected biopolymers. Finally, beta cells in suspension were centrifuged to form controlled numbers of cell layers on films for future work determining the mass transport limitations in the adhered tissue constructs.

Primary monolayer cell culture of bovine parathyroids: Effects of calcium, isoproterenol and growth factors

Cell aggregates of bovine parathyroid tissue were prepared by limited collagenase digestion and placed in culture in Weymouths MB752/1 (calcium = 3.3 mg/100 ml) containing 5% fetal bovine serum and supplemented with insulin alone, or insulin, hydrocortisone, transferrin and epidermal growth factor. Only insulin was required for the maintenance of PTH secretion over a 9-day period. The cell aggregates spread to form monolayer in 3-5 days. The majority of the cells in monolayer were polygonal with well-defined borders. Nuclei were round and the cytoplasm was free of vacuoles. Cell cultures responded to secretory stimulation by low calcium or by isoproterenol with increases in the secretion of PTH and SP-1. At low calcium, about 18% of both the cellular PTH and SP-1 was secreted per hour, and up to 50% of the cell content of these proteins was released per hour upon stimulation by isoproterenol and low calcium combined. The responses to calcium and isoproterenol decreased as a function of time in culture, and calcium responses often disappeared completely by 10 days of culture. When cells were cultured in medium containing a higher (5 mg%) than standard concentration of calcium between days 3-6 of culture, the degree of secretory inhibition attainable with high calcium was greater than that of cells cultured in the standard medium. When secreted hormonal peptides were separated by SDS-gel electrophoresis prior to RIA, it was found that the secretion of intact hormone was sensitive to calcium. For every molecule of PTH secreted into the medium, 1.5-2 mole-equivalents of carboxyl fragments were also released. Calcium control of fragment release was not as stringent as that of PTH release.

Reduction of diffusion barriers in isolated rat islets improves survival, but not insulin secretion or transplantation outcome.

For people with type 1 diabetes and severe hypoglycemic unawareness, islet transplants offer hope for improving the quality of life. However, islet cell death occurs quickly during or after transplantation, requiring large quantities of islets per transplant. The purpose of this study was to determine whether poor function demonstrated in large islets was a result of diffusion barriers and if removing those barriers could improve function and transplantation outcomes. Islets were isolated from male DA rats and measured for cell viability, islet survival, glucose diffusion and insulin secretion. Modeling of diffusion barriers was completed using dynamic partial differential equations for a sphere. Core cell death occurred in 100% of the large islets (diameter > 150 μm), resulting in poor survival within 7 days after isolation. In contrast, small islets (diameter < 100 μm) exhibited good survival rates in culture (91%). Glucose diffusion into islets was tracked with 2-NBDG; 4.2 μm/min in small islets and 2.8 μm/min in large islets. 2-NBDG never permeated to the core cells of islets larger than 150μm diameter. Reducing the diffusion barrier in large islets improved their immediate and long-term viability in culture. However, reduction of the diffusion barrier in large islets failed to improve their inferior in vitro insulin secretion compared to small islets, and did not return glucose control to diabetic animals following transplantation. Thus, diffusion barriers lead to low viability and poor survival for large islets, but are not solely responsible for the inferior insulin secretion or poor transplantation outcomes of large versus small islets.

Biosynthesis of proparathyroid hormone and parathyroid hormone: Chemistry, physiology and role of calcium in regulation

The discovery of bovine proparathyroid hormone is reviewed, and recent findings regarding the structure of the prohormone are presented. Sequence analyses on two prohormone preparations have established that the NH2-terminal region-of the bovine prohormone is the hexapeptide, Lys-Ser-Val-Lys-Lys-Arg, attached to the NH*-terminal Ala of the hormone form. The sequence of the COOH-terminal region of the prohormone is not yet known. Our present data on the human prohormone suggest that it consists of a similar NH*-terminal hexapeptide. The newly synthesized peptides are separable from the bulk of the preexisting hormone by either deoxycholate extraction or sucrose gradient centrifugation, which permits studies of the subcellular distribution of the prohormone and hormone; from such studies a proposal is made concerning the intracellular locales of synthesis and conversion of prohormone to hormone. Glands from rats receiving a low calcium intake convert prohormone to hormone more efficiently than do glands from animals receiving a normal high calcium intake. A model depicting the various sites at which calcium may affect the production of hormone is proposed.

Serotonin in trigeminal ganglia of female rodents : Relevance to menstrual migraine

Objectives.—We examined changes in the serotonin system across the estrous cycle in trigeminal ganglia of female rodents to determine which components are present and which are regulated by the variations in levels of ovarian steroids that occur during the estrous cycle.

Biosynthesis of Proparathyroid Hormone and Parathyroid Hormone by Human Parathyroid Glands

Human parathyroid glands obtained at autopsy were incubated with [(3)H]leucine and [(3)H]lysine. After incubation, nonradioactive parathyroid tissue of either human or bovine origin was added. Radioactive parathyroid hormone and proparathyroid hormone were isolated from the gland and medium by organic solvent and salt fractionation, trichloroacetic acid precipitation, Sephadex G-100 gel filtration, and carboxymethyl cellulose column chromatography. The human hormonal peptides were identified in the ion-exchange column eluates by their relatively high levels of radioactivity, their elution positions, and their immunoreactivity to anti-PTH antiserum. The time-course of radioactive amino acid incorporation into these peptides and a brief incubation of the gland with radioactive amino acids, followed by various lengths of incubation with nonradioactive amino acids, indicated that a precursor-product relationship exists for the two peptides. An alternate method for isolation of the hormone and prohormone, which involves separation of peptides by urea-polyacrylamide gel electrophoresis, confirmed the identities of the human parathyroid hormone and proparathyroid hormone.

The migration behavior of proparathyroid hormone, parathyroid hormone, and their peptide fragments during gel filtration☆

Abstract The migration behavior of bovine proparathyroid and parathyroid hormones, as well as several hormonal peptide fragments, has been analyzed by gel filtration on thin-layer plates and by column chromatography. The two hormones migrated appreciably faster than was expected for their molecular weights. Their migration rates decreased with increasing pH and approached values more characteristic of their molecular weights at pH 11.0. Migration rates were the same over a concentration range of 2 × 10 −6 –0.9 × 10 −3 m . These results indicate that parathyroid hormone does not exist in solution as an aggregate at the concentrations used in these experiments. These studies suggest that the two hormones are asymmetrical, unfolded monomers in acid solution which become more folded or globular at alkaline pH values.

Formation of parathormone 8–34 by cathepsin-D digestion of parathormone and its efficacy as a hormone antagonist

It previously has been shown that digestion of bovine parathormone (bPTH) with cathepsin-D results in rapid cleavage of the hormone between Phe34 and Val35 yielding PTH(1-34) and PTH(35-84). Since bPTH also contains a Phe at residue 7 we have conducted additional studies to determine whether cleavage at this position could occur. We have found that following longer incubation periods of hormone and enzyme, 2 additional peptides are generated; PTH(8-34) and PTH(1-7). Time course studies demonstrated that these 2 fragments are formed from the (1-34) peptide generated through the initial cleavage at Phe34-Val35 of PTH. The identification of the bPTH(8-34) was accomplished through amino acid analysis and N-terminal sequencing. bPTH(8-34) behaved as a PTH antagonist in an in vitro mouse calvarial bone resorption assay. Although bPTH(8-34) did not affect the PTH-stimulated cAMP response when added simultaneously with PTH, preincubation of bone cells with this peptide caused desensitization of the PTH-stimulated cAMP response.

Thapsigargin shifts the CA set point of parathyroid cells to lower extracellular [CA]

The hypothesis that cytosolic calcium concentration ([Ca2+cyt]) is the primary regulator of parathyroid hormone (PTH) secretion is supported by a number of studies that show an inverse relationship between them. One agent shown to inhibit PTH secretion is thapsigargin, a sesquiterpene lactone that raises [Ca2+cyt] by inhibiting the Ca-ATPase that pumps Ca2+ from the cytosol into the lumen of the endoplasmic reticulum. Thapsigargin may act on the parathyroid cell other than to inhibit the Ca-ATPase, however, in ways that might also affect PTH secretion. We have tested its effects on functional parameters, such as protein synthesis, the exocytic machinery, and the ability of parathyroid cells to respond to different concentrations of extracellular Ca2+ ([Ca2+ex]). In particular, we have determined whether the inhibition of PTH secretion by thapsigargin is independent of or is modulated by changes in [Ca2+ex]. The results revealed no effects of thapsigargin on protein synthesis or the exocytic mechanisms within 2 h of treatment, and showed that [Ca2+ex] can modulate PTH secretion in the presence of thapsigargin. Its inhibition of PTH secretion, therefore, appears to rest on its ability to shift [Ca2+cyt] to higher levels, but the possibility that it interacts with the Ca receptor has not been eliminated. The results support the hypothesis that the primary regulator of steady-state PTH secretion is [Ca2+cyt].