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  • Author: S J Persaud x
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Antisense oligonucleotide inhibition of gene expression: application to endocrine systems

S J Persaud and P M Jones

Introduction

With the development of the RIA (Yalow & Berson, 1960), endocrinologists entered the mainstream of biomedical research, and over the years they have maintained their position by adapting biochemical and electrophysiological techniques for use in their particular endocrine cell type. Now they have gone one step further and embraced the discipline of molecular biology. Thus, in recent years, endocrinologists have identified genes and mRNAs within their cells by Southern and Northern blotting, transfected cells with cDNAs encoding particular proteins to characterize protein function, and introduced mutant cDNAs into cells to identify sequences of a protein which are necessary for its function. Of particular interest is the antisense oligonucleotide strategy of selectively depleting cells of individual proteins, a technique which has been applied to many non-endocrine cell types (reviewed by Colman, 1990; Neckers & Whitesell, 1993). So far the antisense oligonucleotide approach has not been used widely in endocrine cells,

DOI:
https://doi.org/10.1677/jme.0.0120127
Volume 12: Issue 2,
Pages:
127–130 (4 total)
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Protein kinase C and the regulation of insulin secretion from pancreatic B cells

P. M. Jones, S. J. Persaud, and S. L. Howell

ABSTRACT

Protein kinase C (PKC) has been identified in islets of Langerhans and insulin-secreting tumour cells. Diacylglycerols (DAGs, the endogenous PKC activators) are generated in response to insulin secretagogues, although nutrient and non-nutrient secretagogues generate DAGs of different compositions and of different potencies as PKC activators. Exogenous activators of PKC stimulate insulin secretion from B cells, but attempts to define a physiological role for PKC by using inhibitors of this enzyme have produced ambiguous results. However, in studies using PKC-depleted B cells the loss of PKC activity does not inhibit glucose-induced insulin secretion, but markedly reduces responses to cholinergic agonists. These observations are supported by measurements of PKC activation which suggest that the enzyme is activated by cholinergic agonists, but not by nutrient secretagogues. Currently available experimental evidence therefore suggests that activation of PKC is not essential for nutrient-induced insulin secretion, but is required for the expression of a normal secretory response to cholinergic neurotransmitters.

DOI:
https://doi.org/10.1677/jme.0.0060121
Volume 6: Issue 2,
Pages:
121–127 (7 total)
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Identification and localisation of a type IV cytosolic phospholipase A2 in rat pancreatic β-cells

K J Parker, P M Jones, C H Hunton, S J Persaud, C G Taylor, and S L Howell

ABSTRACT

The liberation of arachidonic acid (AA), by phospholipase A2 (PLA2), is the rate-limiting step in a number of cell signalling pathways. In the pancreatic β-cell, AA itself is thought to participate in the regulation of insulin secretion. Recently a Ca2+-sensitive, AA-selective cytosolic PLA2 (type IV cPLA2) has been isolated from the human monocyte U937 cell line. Although the DNA sequence of this enzyme implies a molecular weight of 85 kDa, the protein migrates with a molecular weight of 100-110 kDa on SDS-PAGE. In many cell types, cPLA2s which are reactive towards antibodies raised against the type IV cPLA2 have been shown to hydrolyse AA from membrane glycerophospholipids. Using a polyclonal antibody raised against a recombinant form of type IV cPLA2, we have detected an immunoreactive protein with a molecular weight of 93·5 kDa in rat islets of Langerhans. Furthermore, we have detected similar immunoreactive proteins in insulin-secreting β-cell lines and have shown co-expression of type IV cPLA2 immunoreactivity and insulin immunoreactivity in rat pancreatic β-cells. Under non-stimulatory conditions the 93·5 kDa immunoreactive protein detected in rat islets of Langerhans was located predominantly in the cytosolic fraction. We have shown that immunoprecipitation of the rat immunoreactive protein from rat islet homogenates significantly decreases the total dithiothreitol/β-mercaptoethanol-insensitive PLA2 activity by 56·4±7% This provides further evidence that the immunoreactive rat protein is a type IV cPLA2 and is responsible for a large component of the PLA2 activity in rat islets of Langerhans. It is possible that, in the rat β-cell, type IV cPLA2 couples the increase in intracellular Ca2+, brought about by insulin secretagogues, to the liberation of AA and the subsequent release of insulin.

DOI:
https://doi.org/10.1677/jme.0.0170031
Volume 17: Issue 1,
Pages:
31–43 (13 total)