Juxtacellular histamine concentration governs histamine release from rat peritoneal mast cells.

Isolated rat peritoneal mast cells release histamine when superfused with isoosmotic salt or sucrose solutions. The release was ascribed by us to an intracellular ion exchange between potassium and histamine at granule sites, resulting from a flux of cytoplasmic potassium across the granules secondary to the disturbance of the 'state of equilibrium' at the cell surface caused by the superfusion (Uvnäs et al. 1989). In the present article is shown that the histamine releasing effect is counteracted by the addition of histamine to the superfusion fluid. The inhibition is concentration-dependent and accompanied by concomitant changes in the potassium efflux. A 50% inhibition of the histamine release requires an external histamine concentration of 40 microM and extrapolation of the equilibrium curve hints at a total inhibition at concentrations around 170 microM. The observations are taken to indicate that reduction of the juxtacellular histamine concentration caused by the superfusion disturbs the histamine equilibrium at the mast cell surface resulting in the activation of the histamine secretory mechanism. In other words, the secretory activity of the mast cell is checked by the juxtacellular concentration of histamine. When the juxtacellular histamine is removed e.g. on isolation procedures, other experimental situations such as superfusion, or by consumption in vivo the mast cell delivers histamine to restore the juxtacellular equilibrium.

The molecular mechanism of nondegranulative release of biogenic amines.

According to current teaching biogenic amines are released by exocytosis, i.e. by evacuation of amine storing vesicles or granules into the extracellular space. The release of transmitter amines is quantal, i.e. occurs in packs of transmitter molecules. These packs are assumed to be identical with vesicle contents, in other words, the smallest releasable quantum equals the amine content of one vesicle. However, there are experimental observations which do not fit in with this version of an exocytotic release theory. Observed quantitative discrepancies could be explained if the release mechanism allowed a fractional release of transmitter amine from several vesicles instead of the total evacuation of a few. The lack of adequate knowledge about the mechanisms of storage of biogenic amines within the vesicles has up til now rendered it difficult to envisage the machinery behind a fractional release of the amine content of a vesicle. In extensive in-vitro studies we have found that the matrices of amine storing granules (i.e. from mast cells, chromaffin cells and nerve terminals) show the properties of weak cation exchanger materials, carboxyl groups serving as amine binding ionic sites. When exposed to cations like sodium and potassium ions, the amines are released from their storage sites according to kinetics characteristic of weak cation exchangers. In vivo, amine release from cat adrenals on splanchnic nerve stimulation also occurs according to ion exchange kinetics. Histamine release from mast cells is considered to occur as the result of degranulation, i.e. the expulsion of histamine storing granules to the extracellular space, a typical example of exocytosis.(ABSTRACT TRUNCATED AT 250 WORDS)

Urinary bladder and urethral responses to pelvic and hypogastric nerve stimulation and their relation to vasoactive intestinal polypeptide in the anaesthetized dog.

The effects on the urinary bladder and urethra of pelvic and hypogastric nerve stimulation and their relation to vasoactive intestinal polypeptides (VIP) were investigated in the anaesthetized dog. Both pelvic and hypogastric nerve stimulation elicited a twofold increase in urinary bladder blood flow and a clear-cut increase in bladder venous effluent VIP concentration. Hypogastric nerve stimulation induced an initial, partly alpha-adrenergic and partly non-adrenergic, non-cholinergic, contraction of the urinary bladder followed by a relaxation. The urethra response was a maintained alpha-adrenergic contraction. Pelvic nerve stimulation elicited a bladder contraction with an initial non-cholinergic peak, whereafter the bladder pressure was maintained at a lower level, an effect which was mainly cholinergic in origin. The urethral response was an initial non-adrenergic, non-cholinergic contraction followed by a maintained cholinergic contractile response. Afferent pelvic nerve stimulation led to an efferent activity that seemed to be a combination of activity in pelvic and hypogastric pathways to the urinary bladder and the urethra. VIP (10 nmol) injected i.v. induced a relaxation of the urinary bladder and the urethra, together with a fall in systemic blood pressure. However, despite high plasma concentrations, no vasodilation was elicited in the urinary bladder. Thus, the main target for the VIP release during pelvic and hypogastric nerve stimulation is probably not the bladder vasculature, but instead perhaps the bladder smooth muscle proper.

Intracellular ion exchange between cytoplasmic potassium and granule histamine, an integrated link in the histamine release machinery of mast cells.

Rat peritoneal mast cells isolated by gradient centrifugation in Percoll were placed between two membrane filters in a Sartorius filter apparatus and superfused with isotonic balanced salt solutions or with deionized isotonic sucrose. Histamine was released according to ion exchange kinetics. Our explanation of the observed phenomena is as follows. The superfusion induces a flow of cytoplasmic K+ ions across the histamine-containing granules, resulting in an ion exchange K+ in equilibrium Hi+ ions at the histamine binding sites. The concomitant equimolar outflow of histamine and potassium is considered to be due to a functional interplay between two histamine pools, a release and a donor pool. As the result of the K+ in equilibrium Hi+ ion exchange at the histamine binding sites of the release pool, these sites become transiently occupied by K+ ions only to be immediately reoccupied by Hi+ ions from the donor pool. The observed equimolar outflows are consistent with a 1/1 molar ratio in the exchange between histamine and potassium ions. The essential role of cytoplasmic potassium in the histamine release mechanism is a new and important observation with possible implications not only as to histamine release in general (including so-called 'spontaneous' histamine release) but also as to the release of biogenic amines and other positively charged substances stored in granules in ionic linkage to the matrix.

Occurrence of an insulin-like peptide in extracts of human nervous tissue.

Peripheral nerves of the cat such as the vagal and the sciatic nerves have been shown to contain a peptide with insulin-like properties. The aim of the present study was to investigate whether insulin-like immunoreactivity (ILI) can be demonstrated in human nervous tissue collected from autopsy material. Biopsies were taken in connection with autopsy from various peripheral nerves and their content of ILI was investigated. ILI in amounts up to about 12 ng g-1 was found in about 30% of all biopsies taken from peripheral nerves. The ILI coeluted with a bovine insulin standard in an HPLC system indicating that it corresponds to a peptide identical with or similar to pancreatic insulin. Autopsy specimens taken from the sciatic nerve of individuals with diabetes type II or from individuals without established diabetes contained similar amounts of ILI.

Catecholamines (CA) and adenosine triphosphate (ATP) are separately stored in bovine adrenal medulla, both in ionic linkage to granule sites, and not as a non-diffusible CA-ATP-protein complex.

On superfusion of chromaffin granules from bovine adrenals with isotonic sodium and potassium salts, catecholamines and ATP were released in parallel and both in accordance with ion exchange kinetics. An artificial model was prepared by mixing a cationic (IRC-50) and an anionic (IR-4B) ion exchanger with COO- and NH+3 groups, respectively, as binding sites. This mixed ion exchanger showed in its storage and release of CA+ and ATP- striking similarities to the chromaffin granules. Within the pH range given for the interior of the granules--5.5-6--the artificial model even stored and released CA+ and ATP- within the same molar ratio as observed for the granules. We hypothesize that the chromaffin granule matrix in its storage and release functions operates as an amphoteric ion exchanger with COO- and NH+3 groups as the binding sites.

Do superfused mast cells release histamine by endogenous cation exchange with potassium ions?

Isolated rat peritoneal mast cells released histamine on superfusion with isotonic salt solutions or isotonic deionized sucrose. The histamine release followed the kinetics of cation exchange characteristic of the release from similarly superfused isolated mast cell granules and histamine charged carboxylic resin IRC-50. The histamine release was accompanied by an efflux of potassium and ascribed to an endogenous cation exchange K+ in equilibrium with Hi+ occurring on the passage of outflowing potassium ions over histamine storing granules.

Release of an insulin-like peptide from perfused extirpated cat legs in response to electrical stimulation of the sciatic and brachial nerves and to administration of ACh, bombesin, oxytocin and glibenclamide.

The vascular system of extirpated cat legs was perfused with Tyrode's solution and insulin-like immunoreactivity (ILI) levels were determined in the perfusate with radioimmunoassay. During unstimulated conditions perfusate levels of ILI were almost undetectable. However, in response to electrical stimulation of the sciatic or brachial nerves (within a wide range of stimuli 5-40 V, 2-20 Hz and 0.2-40 ms) 1-20 ng of ILI was recorded in the perfusate. Blockers of cholinergic and adrenergic transmissions added to the perfusate did not influence the output of the ILI induced by nerve stimulation. Furthermore, after administration of acetylcholine (ACh) (0.1 and 10 micrograms kg-1), oxytocin (0.5 and 5 IU kg-1), glibenclamide (25 and 100 micrograms kg-1) and bombesin (100 and 500 micrograms kg-1) to the cat leg preparation, ILI appeared in the perfusate in amounts similar to those induced by electrical stimulation of the nerves. When subjected to high-performance liquid chromatography (HPLC) the insulin-like peptide detected in the cat leg perfusate following nervous stimulation, or administration of oxytocin and glibenclamide, co-eluted with a bovine insulin standard. We have previously shown that some peripheral nerves of the cat, such as the sciatic, brachial and vagal nerves, contain an insulin-like peptide with HPLC characteristics similar to the bovine insulin standard. It is therefore possible that the insulin-like peptide released from the isolated cat leg preparation by the above-mentioned stimuli derives from this nervous pool of insulin. Alternatively, the insulin-like peptide emanates from the striatal muscles innervated by the sciatic and brachial nerves, since also muscles have been shown to contain an insulin-like peptide.

Cation exchanger properties of isolated rat peritoneal mast cell granules.

The synthetic carboxylic cation exchanger resin Amberlite IRC-50 was charged with histamine by suspension in histamine-containing solution with admixture of [14C]histamine. Mast cell granules were isolated from mast cells suspended in isotonic sucrose. The release of histamine induced from the two materials by superfusion with isotonic NaCl and KCL solutions showed identical kinetics, in accordance with the view that the release of histamine is due to a cation exchange: Na+ (K+) in equilibrium Hi+ at carboxyl groups in the granule heparin-protein complex.

Cation-induced histamine release from a synthetic weak (carboxylic) cation exchanger resin (IRC-50) and from isolated mast cell granules show identical kinetics.

Comparative studies between synthetic weak cation exchanger resins and rat mast cell granules have shown that the cation-induced release of histamine from both materials follows the kinetics characteristic of cation exchange. Since also cation-induced release of amines from chromaffin granules in vitro and chromaffin cells in vivo, as also nerve granules of peripheral and central neurons, run according to cation exchange kinetics, cation exchange might be a general principle in the storage and release of biogenic amines.

The kinetics of adrenal catecholamine secretion elicited by splanchnic nerve stimulation or by Ach is consistent with non-exocytotic, multivesicular release on cation exchange basis.

In eight anaesthetized cats, one dog and one pig the left adrenal was activated during a 5-15-min period by splanchnic nerve stimulation (10-30 V, 0.2-2 ms) at supramaximal frequencies (10-50 Hz) or by i.a. infusion of acetylcholine in high concentration (10(-4) M). The catecholamine (CA) release, as recorded in the adrenal venous outflow, was characterized by a very steep rise to a peak (within less than 10 s), followed by a rapid decline which after 5-10 min continued as a 'steady state' secretion, still above prestimulatory level. The initial release curve satisfied the straight line equation log B = K square root (sigma ml) + log Bmax, shown previously by us to be characteristic of the cation-induced amine release from amine-charged IRC 50 (a synthetic carboxyl cation exchanger resin) and from chromaffin granules in vitro which occurred on superfusion of these materials with isotonic NaCl solution (Uvnäs & Aborg 1984a). The initial CA-release, which depending on the intensity of the stimulus amounted to between 0.1 and approximately 5% of the adrenal CA content is suggested to reflect the rapid depletion of a CA pool for immediate release composed of granules 'lined up' for secretion adjacent to the plasma cell membrane. On depolarization of this membrane the granules are assumed to become attached to it and CA release to occur as a cation exchange, between CA+ in the granule matrix and Na+ in the plasma or possibly K+ in the cytoplasm. The transition from depletion to 'steady state' phase is assumed to reflect resynthesis or other compensatory refilling of the releasable depot evoked by its depletion. Cation exchange is suggested to be a general principle in the release of biogenic amines, including transmitter amines and other co-stored charged substances, e.g. polypeptides.

Cation exchange--a common mechanism in the storage and release of biogenic amines stored in granules (vesicles)? III. A possible role of sodium ions in non-exocytotic fractional release of neurotransmitters.

The matrices of the amine storing granules in mast cells, chromaffin cells and noradrenergic nerves show properties reminiscent of cation exchanger materials. In vitro, the amines are released from their granule storage sites on exposure of the granules to cations, e.g. sodium ions. The proposal is made that also in vivo the release of transmitter amines is the result of cation exchange Amine+ in equilibrium Na+ ions and that the release of transmitter amines occurs as a nonexocytotic fractional release engaging multiple granules instead of exocytotic emptying of a few. Some physiological and pharmacological implications of a fractional transmitter release are discussed.

Cation exchange--a common mechanism in the storage and release of biogenic amines stored in granules (vesicles)? II. Comparative studies on sodium-induced release of biogenic amines from the synthetic weak cation-exchangers Amberlite IRC-50 and duolite CS-100 and from biogenic (granule-enriched) materials.

Superfusion of phenylethylamine-, noradrenaline- or histamine-charged weak (carboxyl) cation-exchangers (IRC-50 and Duolite CS-100) with isotonic NaCl caused a release of the amines. Similarly, bovine chromaffin granules and nerve granule preparations from bovine splenic nerve, rat vas deferens and rat corpus striatum released their amine(s) upon superfusion with the same solution. The courses of release from the synthetic and biogenic materials showed very similar characteristics and fitted the same exchange equations. The observations support the view that the matrices of the biogenic amine-storing granules have the properties of weak cation-exchanger materials with carboxyls as the cation-binding groups, and that the NaCl-induced release of the biogenic amines is due to cation exchange (Na+ in equilibrium Amine+). The possibility that amine release in vivo is based on cation exchange is discussed.