The IP3‐sensitive calcium store of HIT cells is located in a surface‐derived vesicle fraction

Abstract

Electron microscopic and biochemical techniques were used to study the cellular localization of the ATP‐dependent, IP3‐sensitive, Ca2+ store in the glucose‐ and phosphatidylinositol(PI) agonist‐sensitive hamster insulinoma cell line HIT‐T15. Scanning electron microscopy revealed conspicuous shape changes of the microvilli following stimulation of these cells with bombesin or thapsigargin. These changes closely resemble those previously shown to accompany stimulation of hexose transport in adipocytes with insulin [J. Cell. Physiol. 142 (1990) 1‐14]. Using a hydrodynamic shearing technique for the isolation of microvilli, two cell surface‐derived vesicle fractions were prepared containing 80% of the total cellular Ca2+‐storing activity. In contrast, subcellular fractionation using normal homogenization with a glass/teflon homogenizer yielded the well‐known distribution of the Ca2+‐storing activity which is then predominantly recovered within the microsomal fraction. The surface‐derived vesicle fraction was clearly distinguished from the microsomal fraction by its high content of Na+/K+‐ATPase and an immunoreactive fragment of the GluT‐1 glucose transporter isoform which both are not detectable in the microsomal fraction isolated from homogenates from sheared cells. The Ca2+ uptake properties of the cell surface‐derived vesicle fractions including the vanadate, A23187, and thapsigargin sensitivity were found to be identical with those described for the microsomal Ca2+ stores of various cell types. Inositol 1,4,5‐trisphosphate (IP3) at 1 μM induced a maximal release of 35–40% of the stored Ca2+ from these vesicles.

Dopamine (D2) receptor regulation of intracellular calcium and membrane capacitance changes in rat melanotrophs.

“…Interestingly calcium released by IP3 stimulated higher secretion rates. These secretion rates are, however, similar to secretion rates in gonadotrophs (Tse, Tse, Almers & Hille, 1993) and pancreatic fl-cells (Ammala et al 1993) (Dunlop & Larkins, 1988), hepatocytes (Rossier, Bird & Putney, 1991), and insulin-secreting cells (Lange & Brandt, 1993) have suggested that some calcium stores are near the plasma membrane. The observation of IP3 receptors on secretory vesicles in chromaffin cells and insulin-secreting cells suggest an even more direct colocalization of calcium release sites with secretory vesicles (Yoo & Albanesi, 1990;Blondel et al 1994 Yamamoto et al (1987) had previously shown that the total amount of secretion could be increased maximally 2-fold with 2 JiM cAMP at about 4/M Ca!+ in electropermeabilized melanotrophs.…”

Cellular target of weak magnetic fields: ionic conduction along actin filaments of microvilli

“…Because of tighter binding of divalent cations to fixed charge centers, divalent cation conduction is more restricted. increases following receptor-mediated disorganization of the microvillar bundle structure (40,43,44).…”

Microvillar Ca++ signaling: A new view of an old problem

“…Using a shearing protocol identical to that used for the preparation of glucose transporter-containing membrane fractions from adipocytes, Ca ϩϩ -storing vesicles were isolated from the surface of a hamster insulinoma cell line (HIT; Lange and Brandt, 1993a) and from hepatocytes . The Ca ϩϩ storage properties, the experimental conditions for ATP-dependent Ca ϩϩ uptake as well as the inhibitor spectrum of these vesicle fractions were identical to those of the microsomal systems from various cell types generally assigned to the endoplasmic/sarcoplasmic reticulum Ca ϩϩ ATPases.…”