Identification of the mechanism responsible for the increased fibrin specificity of TNK-tissue plasminogen activator relative to tissue plasminogen activator.

TNK-tissue plasminogen activator (TNK-t-PA), a bioengineered variant of tissue-type plasminogen activator (t-PA), has a longer half-life than t-PA because the glycosylation site at amino acid 117 (N117Q, abbreviated N) has been shifted to amino acid 103 (T103N, abbreviated T) and is resistant to inactivation by plasminogen activator inhibitor 1 because of a tetra-alanine substitution in the protease domain (K296A/H297A/R298A/R299A, abbreviated K). TNK-t-PA is more fibrin-specific than t-PA for reasons that are poorly understood. Previously, we demonstrated that the fibrin specificity of t-PA is compromised because t-PA binds to (DD)E, the major degradation product of cross-linked fibrin, with an affinity similar to that for fibrin. To investigate the enhanced fibrin specificity of TNK-t-PA, we compared the kinetics of plasminogen activation for t-PA, TNK-, T-, K-, TK-, and NK-t-PA in the presence of fibrin, (DD)E or fibrinogen. Although the activators have similar catalytic efficiencies in the presence of fibrin, the catalytic efficiency of TNK-t-PA is 15-fold lower than that for t-PA in the presence of (DD)E or fibrinogen. The T and K mutations combine to produce this reduction via distinct mechanisms because T-containing variants have a higher K(M), whereas K-containing variants have a lower k(cat) than t-PA. These results are supported by data indicating that T-containing variants bind (DD)E and fibrinogen with lower affinities than t-PA, whereas the K and N mutations have no effect on binding. Reduced efficiency of plasminogen activation in the presence of (DD)E and fibrinogen but equivalent efficiency in the presence of fibrin explain why TNK-t-PA is more fibrin-specific than t-PA.

In situ imaging of agonist-sensitive calcium pools in AR4-2J pancreatoma cells. Evidence for an agonist- and inositol 1,4,5-trisphosphate-sensitive calcium pool in or closely associated with the nuclear envelope.

The activation of phospholipase C by hormones and neurotransmitters activates a complex combination of Ca2+ release and accumulation by intracellular organelles. Previously, we demonstrated that, in some cell types, the fluorescent Ca2+ indicator, fura-2, can be loaded into intracellular, agonist-sensitive Ca2+ pools (Glennon, M. C., Bird, G. St. J., Kwan, C.-Y., and Putney, J. W., Jr. (1992) J. Biol. Chem. 267, 8230-8233). In the current study, we have attempted to exploit this phenomenon by employing digital fluorescence imaging of compartmentalized fura-2 to investigate the localization and function of the major intracellular sites of Ca2+ regulation in AR4-2J pancreatoma cells. By judicious use of a surface receptor agonist together with the Ca(2+)-ATPase inhibitor, thapsigargin, cellular regions were identified whose behavior indicates that they contain the sites of agonist- and inositol 1,4,5-trisphosphate-mediated intracellular Ca2+ release. These regions were located throughout the cell and may include the nuclear envelope. They were distinct in locus and behavior from two other regions, which counterstained with fluorescent markers for nuclei and mitochondria. Fura-2 in mitochondrial regions reported low resting levels of [Ca2+], and revealed that organelles in these regions accumulate and retain Ca2+ after agonist activation. These findings demonstrate that fluorescent Ca2+ indicators can be employed to directly monitor changes in [Ca2+] in the major Ca(2+)-regulating organelles, and provide the first in situ visualization and localization of the major sites of Ca2+ regulation in cells.

Persistent inhibition by inositol 1,4,5-trisphosphate of oxalate-dependent 45calcium accumulation in permeable guinea-pig hepatocytes.

Guinea-pig hepatocytes whose plasma membranes were rendered permeable by treatment with saponin, accumulated 45calcium in the presence of potassium oxalate and ATP. The uptake was linear with time for up to one hour when high-capacity EGTA buffers were used (5mM). In the presence of a supra-maximal concentration of inositol 1,4,5-trisphosphate, under conditions minimising metabolism of this calcium-mobilising messenger, 45calcium accumulation was inhibited by about 40% for a period of one hour. Electron microscopic examination of the cells, revealed the presence of electron dense precipitates. Electron microprobe analysis of the precipitates indicated that they constituted the majority of the oxalate-dependent calcium uptake. The precipitates were located throughout the non-nuclear regions of the cells. Cells treated with inositol 1,4,5-trisphosphate contained fewer precipitates, but high cell-to-cell variability prevented conclusions as to the precise location of the pool sensitive to inositol 1,4,5-trisphosphate. These results support the previous contention that a fraction of endoplasmic reticulum is completely emptied of calcium by maximal concentrations of inositol 1,4,5-trisphosphate, while another fraction is insensitive to this action. In addition, these findings indicate that the pool of intracellular calcium on which inositol 1,4,5-trisphosphate acts is oxalate-permeable, and that the calcium-releasing action of inositol 1,4,5-trisphosphate does not desensitise within one hour.

Phorbol ester-induced protein secretion in rat parotid gland. Relationship to the role of inositol lipid breakdown and protein kinase C activation in stimulus-secretion coupling.

When added to rat parotid gland slices incubated in vitro, 4 alpha-phorbol-dibutyrate (PDBu) induced a dose-dependent increase in protein secretion, but did not affect membrane permeability to K+ (as determined by 86Rb efflux). The response to PDBu was unaffected by the removal of extracellular Ca2+ and was not markedly potentiated by incubation with the phosphodiesterase inhibitor, methylisobutylxanthine. PDBu did not activate phospholipase C breakdown of inositol lipids as shown by a failure to increase formation of soluble inositol phosphates. When applied in combination with the Ca2+ ionophore, ionomycin, a secretory rate was obtained that was greater than the predicted sum of rates obtained when the two drugs were given alone. These results, when taken with the reported results of others, are consistent with an action of PDBu in activating protein kinase C and suggest that this enzyme plays an important role in the pathway linking receptor activation to protein secretion, but not K+ flux, in the parotid gland.

The Whitaker Index of Schizophrenic Thinking (WIST) and thirteen systems for diagnosing schizophrenia.

Developed the present study in conjunction with a project that evaluated the psychiatric diagnosis of schizophrenia (Landmark, 1982; Landmark & Leslie, 1982). This design, which included 13 different interview systems for diagnosing schizophrenic disorders, allowed for an in-depth analysis of the Whitaker Index of Schizophrenic Thinking (WIST). Eighty-five outpatients who were receiving long-term chemotherapy for schizophrenia at the Moditen Clinic of London Psychiatric Hospital were reassessed individually using each of the 13 systems, following a clinical interview format, and the WIST. Major statistical analysis evaluated the concurrent validity and discriminatory efficiency of the WIST in comparison to the 13 systems. The WIST was found to show a consistently high agreement with each of the 13 systems for an average WIST discriminatory efficiency of 70%. The WIST is suggested as a practical, easily administered, diagnostic tool that performs well in comparison to the interview systems.

Calcium pools in saponin-permeabilized guinea pig hepatocytes.

The plasma membranes of isolated guinea pig hepatocytes were made permeable with saponin. The cells were then suspended in a medium resembling cytosol in which the level of ATP was kept constant with an ATP-regenerating system. Intracellular ATP-dependent 45Ca and 40Ca sequestration was then followed at various concentrations of Ca2+ in the medium. It was found that ATP-dependent Ca uptake could be divided into two mechanisms: a low affinity high capacity uptake sensitive to 2,4-dinitrophenol (DNP) and oligomycin, thought to be mitochondrial, and a low capacity high affinity uptake, which was insensitive to DNP and oligomycin, thought to be mainly endoplasmic reticulum (ER). The threshold for ATP-dependent Ca uptake by the latter pool was about 20 nM Ca2+. The process had an EC50 value of 0.3 microM (for 45Ca) and a capacity of 2.7 nmol/45Ca/mg of protein. The "ER" mechanism also had a high affinity for ATP (EC50, about 43 microM). There was no significant accumulation of Ca by the postulated mitochondrial pool until the [Ca2+] of the medium was greater than 1 microM. The concentration of Ca2+ in the cytosol of normal unstimulated hepatocytes was estimated from measurements of phosphorylase a activity to be about 0.18 microM. At this [Ca2+], the ER pool of the saponin-treated hepatocytes accumulated Ca but there was no evidence of any Ca uptake into the "mitochondrial" pool. This suggests that most of the exchangeable Ca in a normal cell may be in DNP and oligomycin-insensitive pools (presumably the ER or possibly the plasma membrane) and suggests that these pools are likely to be involved in the increase in cytosolic [Ca2+] which occurs after stimulation by Ca-mobilizing hormones.

Ionic mechanisms in secretagogue-induced morphological changes in rat parotid gland.

When 10(-5) M carbachol was added to parotid tissue slices incubated in buffer containing Ca++, watery vacuoles were formed in the cells. The percent volume density of vacuoles, as measured from 0.5-micron sections, increased from 0.64 +/- 0.15 SE (n = 7) to 3.09 +/- 0.99 (n = 5) in 10 min and, finally, to 7.27 +/- 1.88 (n = 4) in 30 min. In electron micrographs, most of the vacuoles appeared to arise from a location near the Golgi apparatus. Condensation of nuclear chromatin and a conformational change in mitochondria were also noted immediately after stimulation. The percent volume density values returned to basal levels with the addition of either 5 mM EGTA or 10(-6) M atropine after the addition of carbachol. Nuclei and mitochondria returned to normal configurations. In the presence of either 1 mM ouabain or high K+, or in the absence of added Ca++, carbachol failed to induce vacuole formation. However, low Na+ medium did not prevent the formation of vacuoles due to carbachol. Ultrastructural changes in nuclei and mitochondria were consistently associated with the appearance of vacuoles. Since both high K+ and ouabain blocked vacuole formation, it is unlikely that Na+ or K+ movements were important for the response. Rather, receptor-activated Ca++ influx, which is likely to be inhibited by depolarizing agents (such as high K+ or ouabain), is probably the more important factor in vacuole formation and other concomitant ultrastructural changes.

Actions of ionomycin in rat parotid gland.

The effects of ionomycin (SQ 23,377), a carboxylic acid Ca-ionophore, on the rat parotid acinar cell were investigated. Ionomycin stimulated 86Rb efflux from parotid slices and was substantially more potent and efficacious than the Ca-ionophore, A-23187. The release of 86Rb was dependent on the concentration of ionomycin and of Ca. Ionomycin also stimulated 22Na uptake and 3H-protein secretion, but did not stimulate the incorporation of 32PO4 into phosphatidylinositol. These observations are consistent with an action of ionomycin in increasing cytosolic Ca by acting as an ionophore and not involving endogenous receptors. Pretreatment with ionomycin inhibited the transient, Ca-independent responses to carbachol or physalaemin. When ionomycin was added to parotid cells pre-equilibrated with 45Ca, a net loss of radiocalcium was observed. These observations suggest that ionomycin can release the receptor-regulated cellular Ca pool. Morphological studies did not reveal any nonspecific deleterious effects in the cells after incubation with 2.67 microM ionomycin.

Muscarinic and alpha-adrenergic stimulation of Na and Ca uptake by dispersed lacrimal cells.

Rat lacrimal gland acinar cells were isolated and observed to be physiologically stable for several hours of incubation in vitro. With a double-isotope technique, it was found that carbachol and epinephrine stimulated the uptake of 22Na and 45Ca by lacrimal cells. These respnses were maximal at agonist concentrations of 10(-5) M and were blocked by atropine and phentolamine, respectively. It is concluded that muscarinic and alpha-adrenergic receptor activation increase the membrane permeability of the lacrimal gland acinar cell to Na and Ca, ions that may be important in the secretion of water by the lacrimal gland.

Stimulus-secretion coupling in the rat lacrimal gland.

The role of calcium in stimulus-secretion coupling in the lacrimal gland was investigated. Rat exorbital lacrimal gland slices released peroxidase into the incubation medium after muscarinic or alpha-adrenergic receptor stimulation. In both cases, secretion appeared to involve exocytosis as revealed by electron microscopy. Secretion due to the muscarinic agonist carbachol was inhibited by Ca omission and by lanthanum. Secretion due to the alpha-adrenergic agonist phenylephrine was inhibited by Ca omission at 10(-5) M concentration but not at 10(-4) M concentration. The effects of 10(-4) M phenylephrine could be blocked by lanthanum or by reduction of extracellular Na. Both agonists stimulated 45Ca efflux, but phenylephrine was less efficacious. These results suggest that the agonists may cause exocytosis by stimulating influx of Ca or release of cellular Ca. Sodium may play a role in the Ca-release mechanism.

Calcium and the control of potassium efflux in the sublingual gland.

The effects of cholinergic stimulation on K efflux from rat sublingual gland slices was investigated. The sublingual gland slices appeared stable on incubation as indicated by electrolyte content and ultrastructural analysis. Carbachol induced a biphasic increase in release of 86Rb (an index of K efflux); a transient phase lasting 2--4 min was followed by a sustained (or slowly falling) phase. Both phases of the response were blocked by atropine, but only the sustained phase was blocked by omission of Ca or by the addition of LaCl3. The divalent cationophore A-23187 produced a Ca-dependent release of 86Rb. Substance P stimulated a biphasic release of 86Rb, similar to that obtained with carbachol, but epinephrine did not. The response to substance P demonstrated a Ca dependence similar to that of carbachol. When a transient response to carbachol was elicited, no transient response to carbachol was elicited, no transient response to substance P could be obtained. This suggests that the receptors for these agonists may reside in the same cells. Also, the magnitude of the responses suggests that most of the affected cells are probably the mucous elements of the sublingual gland.

Is calcium the final mediator of exocytosis in the rat parotid gland?

Rat parotid slices were partially depleted of cellular Ca by long (80-100 minute) incubations in media containing no added Ca and 5mM ethylene glycol bis(beta-aminoethyl ether)-N,N'-tetraacetic acid (EGTA). This treatment inhibited the secretory response (release of alpha-amylase) both to isoproterenol and to dibutyryl cyclic adenosine 3':5'-monophosphate. The isoproterenol-stimulated synthesis of cyclic adenosine 3':5'-monophosphate was inhibited by depletion of Ca but not to an extent sufficient to explain the effects of depletion of Ca on secretion. Isoproterenol did not affect influx of 45Ca but stimulated efflux of 45Ca suggesting release of Ca from intracellular stores. Isoproterenol caused vacuolation of the Golgi region and (in high concentration) enhanced the release of 86Rb, responses which are both believed to be mediated by an increase in cytoplasmic Ca concentration. The results of these experiments suggest that isoproterenol acts to increase the tissue level of cyclic adenosine 3':5'-monophosphate which in turn acts to release Ca from intracellular stores. The rise in intracellular Ca concentration is believed to mediate exocytosis.

alpha-Adrenergic, beta-adrenergic and cholinergic mechanisms for amylase secretion by rat parotid gland in vitro.

1. Rat parotid gland slices, incubated in a balanced, buffered salt solution, were found to be physiologically stable for up to 2 hr with respect to O2 consumption, water content, extracellular space and cation content. 2. The slices could be stimulated to secrete amylase by activation of alpha-adrenergic, beta-adrenergic or muscarinic cholinergic receptors. 3. The secretion elicited through all three receptors appeared to involve exocytosis as revealed by electron microscopy. 4. The beta-agonist, isoprenaline, increased tissue content of cyclic adenosine 3',5'-monophosphate (cyclic AMP); alpha-adrenergic and cholinergic agents had no effect on the level of this cyclic nucleotide. 5. Secretion via cholinergic or alpha-adrenergic mechanisms required extra-cellular calcium; the beta-adrenergic mechanism did not. 6. It was concluded that stimulation of rat parotid cells activates distinctly separate pathways leading ultimately to exocytosis, one pathway involving cyclic AMP, and the other, external Ca2+ ion.