Function of SERCA mediated calcium uptake and expression of SERCA3 in cerebral cortex from young and old rats.

Previous work on peripheral sympathetic neurons indicated that a decline in sarco/endoplasmic reticulum calcium ATPase (SERCA) function occurs with advancing age. Therefore, an age-related decline in mechanisms controlling intracellular calcium homeostasis could contribute to altered neuronal function and/or degeneration. In this study we sought to extend the findings on peripheral neurons and to detect possible age-related declines in SERCA function and expression of SERCA3 in central neurons from cerebral cortex from young (6-month) and old (20-month) rats. Functional studies compared ATP-dependent 45Ca(2+)-uptake into microsomes and plasma membrane vesicles (PMVs). We and found no significant difference in 45Ca(2+)-uptake between microsomes or PMVs between young and old animals. On the other hand expression of SERCA3 mRNA in rat cerebral cortex showed a significant decline with advancing age. However, comparison of SERCA3 protein content did not reveal a corresponding decline; implying that SERCA mRNA turnover rates may be greater in the younger group. Although the present work with rat cerebral cortex does not indicate an age-related decline in SERCA function, previous work from our laboratory on sympathetic nerves and by others on the hippocampus indicate such a decline. In light of our previous and current studies, aging may affect calcium homeostatic mechanisms in central and peripheral autonomic neurons differently.

N-acetylneuraminic acid (NANA) stimulates in situ cyclic AMP production in tentacles of sea anemone (Aiptasia pallida): possible role in chemosensitization of nematocyst discharge.

Cnidocytes, the stinging cells of cnidarians, optimally discharge nematocysts in response to combined physical contact and stimulation of specific chemoreceptors. In the tentacles of certain sea anemones, the primary chemoreceptors bind N-acetylated sugars, such as N-acetylneuraminic acid (NANA). Sensitization with NANA predisposes contact-sensitive mechanoreceptors (CSMs) to trigger discharge in response to physical contact. In the ectoderm of sea anemone tentacles, cnidocyte/supporting cell complexes (CSCCs) control and trigger nematocyst discharge. Previous findings have implicated cyclic AMP (cAMP) as a second messenger in NANA-sensitized nematocyst discharge. However, no reports have directly demonstrated that the cAMP content of tentacles changes in response to NANA stimulation. We now show that NANA elevates in situ cAMP levels in a dose-dependent manner in the ectoderm of tentacles from the sea anemone Aiptasia pallida. However, the endoderm of tentacles shows no detectable cAMP response to NANA. The effect of NANA on the cAMP content of the ectoderm is biphasic. Micromolar NANA increases the in situ cAMP level, with a maximal response occurring at 1.8x10(-5)mol x l(-1) NANA. At higher NANA concentrations, the cAMP content decreases to that of controls. Because the cAMP dose/response curve to NANA coincides precisely with the dose/response curves of NANA-sensitized nematocyst discharge and nematocyst-mediated adhesive force, a second-messenger role for cAMP in NANA-sensitized nematocyst discharge is strongly suggested. The addition of isobutyl-1-methylxanthine (IBMX) to the medium with sea anemones increases tissue cAMP levels both in the absence and in the presence of NANA. However, anesthetizing anemones in sea water containing high levels of Mg(2+) blocks the NANA-stimulated cAMP response of the ectoderm. In addition, our results suggest that NANA-stimulated cAMP may activate endogenous cAMP-dependent protein kinase (PKA) in broken cell preparations of tentacles. Thus, NANA-stimulated cAMP may function as a second messenger in the NANA chemosensory signaling pathway controlling nematocyst discharge.

Enzymatic characterization of the major phospholipase A2 component of sea anemone (Aiptasia pallida) nematocyst venom.

The purified beta phospholipase A2 (PLA2; EC 3.1.1.4) (PLA2) from sea anemone (Aiptasia pallida) nematocysts is larger and more labile than other known venom PLA2s. In common with all other known venoms and most secretory PLA2s, the beta PLA2 requires mM Ca2+ for optimal activity and is surface-activated by aggregated lipids such as mixed micelles of detergent and phospholipid. The beta PLA2 exhibits an unusually steep and narrow pH optimum of activity at pH 7.7. The effects of changes in pH on the activity of the enzyme suggest that the active site contains functional groups having a pKs of about 7.0 and 8.0. The effects of temperature on beta PLA2 activity show a marked decrease in the energy of activation above the pre-transition temperature, suggesting that the enzyme "melts" both fatty chains in order for catalysis to occur.

Portuguese Man-of-war (Physalia physalis) venom induces calcium influx into cells by permeabilizing plasma membranes.

Portuguese Man-of-war (Physalia physalis) nematocyst venom dose-dependently stimulates calcium (45Ca(2+)) influx into L-929, GH(4)C(1), FRL, and embryonic chick heart cells. Venom-induced calcium influx is not blocked by ouabain, vanadate, nor organic calcium channel blockers, but is blocked by transition metal cations, such as lanthanum and zinc. Venom-induced calcium influx is accompanied in a dose-dependent manner by the release of intracellular lactate dehydrogenase, indicating a loss in plasma membrane integrity and cytolysis. Concentrations of zinc that block 45Ca(2+) influx also block lactate dehydrogenase release. Lanthanum, which also blocks 45Ca(2+) uptake, does not neutralize the cytolytic activity of the venom, but rather inhibits the venom's cytolytic action at the level of the target cell plasma membrane. Our findings indicate that Man-of-war venom causes an influx of calcium into several different cells types, not just those of the cardiovascular system, and this influx likely occurs by permeabilizing the plasma membranes of cells.

The effect of Portuguese Man-of-war (Physalia physalis) venom on calcium, sodium and potassium fluxes of cultured embryonic chick heart cells.

Portuguese Man-of-war venom markedly increases calcium (45Ca2+) influx into primary, cultured, embryonic chick heart cells. This action is dose-dependent, but is unaffected by organic calcium blockers (diltiazem, verapamil, nifedipine, nimodipine and mibefradil). On the other hand, certain trivalent (La3+, Gd3+) and divalent (Zn2+, Ni2+, Cu2+, Mn2+) metals inhibit venom-induced calcium influx. Sodium (22Na+) influx into chick heart cells is also significantly increased by Man-of-war venom. Flecainide does not block venom-induced sodium influx. The efflux of the potassium analogue, 86Rb+, from heart cells is also significantly increased by the venom. The venom, however, has little or no effect on rubidium (86Rb+) or 2-deoxy-D-[2-3H] glucose influx.

Purification and partial characterization of the phospholipase A2 and co-lytic factor from sea anemone (Aiptasia pallida) nematocyst venom.

Functional nematocysts of one specific morphological class, the penetrant microbasic mastigophores, were isolated from the sea anemone, Aiptasia pallida. These nematocysts contain a multicomponent venom composed of several proteins, including those with neurotoxic, hemolytic, and lethal activities. Hemolytic activity is produced by at least three synergistic venom proteins. One of these proteins is identified as a phospholipase A2 (EC 3.1.1.4) which exists in two isozymic forms, alpha and beta, with molecular weights of 45,000 and 43,000, respectively. The beta isozyme has been purified to homogeneity. It is a single-chained glycoprotein with an isoelectric point (pI) of 8.8 and represents 70% of the phospholipase activity of the venom. The activity of the beta isozyme is relatively labile and is inactivated by 3.5 M urea or by heating at 45 degrees C. It is most stable at pH 4.0 and loses 50% of its activity at pH values below 3.5 and above 8.0. A second venom protein has also been purified. It is essential for the hemolytic activity of the venom and is termed co-lytic factor (CLF). It is a monomeric glycoprotein having a pI of 4.5. CLF has a molecular weight of approximately 98,000, a sedimentation coefficient of 4.8 S, and is prolate in shape, having a frictional ratio of about 1.6. CLF constitutes about 1.25% of the total venom protein and is assayed by reversing fatty acid inhibition of the venom hemolysis activity.

Efferent Mechanisms of Discharging Cnidae: II. A Nematocyst Release Response in the Sea Anemone Tentacle.

Feeding behavior in cnidarians is a sequence of coordinated responses beginning with nematocyst discharge. The nematocyst response produces prey capture by envenomating prey and attaching prey to the tentacle. The strength of attachment of discharged nematocysts to the tentacle is termed intrinsic adherence and is calculated from measurements of adhesive force. Following prey capture, the feeding response involves movement of the tentacles toward the mouth and mouth opening. For ingestion to occur, nematocysts attaching the prey to the tentacles must be released from the tentacle. A nematocyst release response has been proposed, but never documented nor measured. Our criterion for a nematocyst release response is that the intrinsic adherence of discharged nematocysts must decrease to zero. The unit of nematocyst discharge in sea anemone tentacles is the cnidocyte/ supporting cell complex (CSCC). The nematocyst response includes nematocysts discharged from Type C CSCCs by physical contact alone and nematocysts discharged from the more numerous Type B CSCCs that require both chemosensitization and physical contact. We identify two prey-derived substances, N-acetylneuraminic acid (NANA) and glycine, both of which chemosensitize nematocyst discharge from Type B CSCCs at low concentrations. At higher concentrations NANA stimulates the release response of Type Cs, and glycine stimulates the release response of Type Bs.

Efferent Mechanisms of Discharging Cnidae: I. Measurements of Intrinsic Adherence of Cnidae Discharged From Tentacles of the Sea Anemone, Aiptasia pallida.

Two kinds of cnida predominate in the tentacles of the acontiate sea anemones: spirocysts and microbasic mastigophore nematocysts. These cnidae discharge in response to appropriate mechanical and chemical stimulation. In this paper, we calculate the strengths of attachment between the tentacle and the capsules (= "tentacle adherence") of discharged spirocysts and mastigophores by measuring adhesive force and by determining the numbers of spirocysts and mastigophores discharged onto targets under conditions where the attachment of everted cnida tubules to the target (= "cnida adhesion") exceeds tentacle adherence. Under these conditions, the average contribution of individual cnidae to adhesive force is called the intrinsic adherence. The intrinsic adherence is a measure of the average frictional force required to dislodge the capsule of individual discharged cnidae from the tentacle. The intrinsic adherence of discharged mastigophores varies inversely, from 0.45 to 0.15 mgf (4.41 to 1.47 µN), with the number of discharged mastigophores. The larger values characterize mastigophores discharged by mechanically triggering nonchemosensitized tentacles, whereas the lower values characterize the intrinsic adherence of mastigophores discharged from chemosensitized tentacles. In contrast, the intrinsic adherence of discharged spirocysts is very low to insignificant. Thus, by comparison to mastigophores, spirocysts contribute little, if any, to adhesive force, and, by inference, do not directly secure captured prey to the tentacle. Our measurements indicate that penetrable prey are primarily secured to the tentacle by discharged mastigophores and by the inherent stickiness of the tentacle surface.

Evidence for calcium channels involved in regulating nematocyst discharge.

In the tentacles of sea anemones, nematocyst discharge is regulated by cnidocyte/supporting cell complexes (CSCCs) of which three functional types have been identified: A, B and C. Type A CSCCs respond to contact by vibrating targets. Types B and C CSCCs respond to contact by static targets. Whereas type C CSCCs respond to contact alone, type B CSCCs require that surface chemoreceptors bind ligands before becoming responsive. Reducing Ca2+ levels in artificial seawater to below 1 mM inhibits discharge from each type of CSCC. The calcium channel inhibitors, nifedipine or verapamil, selectively inhibit discharge from type B CSCCs. The calcium channel activator, Bay K-8644, mimics the biphasic dose response of type B CSCCs to natural chemosensitizers such as N-acetylated sugars. Discharge from type A CSCCs is unaffected by inhibitors of L-type calcium channels, but is selectively inhibited by the aminoglycoside antibiotics, gentamicin and streptomycin. While each type of CSCC requires extracellular calcium, the calcium channels employed may vary according to CSCC type.

Receptors for N-acetylated sugars may stimulate adenylate cyclase to sensitize and tune mechanoreceptors involved in triggering nematocyst discharge.

In fishing tentacles of sea anemones, cnidocyte/supporting cell complexes (CSCCs) trigger the discharge of nematocysts following stimulation by swimming prey of specific mechanoreceptors and chemoreceptors located on the supporting cells. Two types of mechanoreceptors have been identified: a contact-sensitive mechanoreceptor (CSM), and a vibration-sensitive mechanoreceptor (VSM). The CSMs become predisposed to initiate nematocyst discharge into static (i.e., nonvibrating) test probes in the presence of submicromolar free and conjugated N-acetylated sugars, a process referred to as sensitization. In seawater, the VSMs cause maximal discharge in response to test probes vibrating at 30, 50-55, and 75 Hz, whereas in the presence of submicromolar N-acetylated sugars the VSMs cause maximal discharge into test probes vibrating at 5, 15, 30, and 40 Hz, a process referred to as tuning. Tuning of the VSMs is accompanied by elongation of the stereocilium bundles comprising the VSMs. We report that dibutyryl cyclic-AMP sensitizes CSMs and tunes VSMs to the lower frequencies of 5, 15, 30, and 40 Hz, while cyclic-AMP has no such effects. Endogenous adenylate cyclase activity at the apical plasma membrane of the supporting cells is detectable by cytochemical methods in the presence of N-acetylated sugars but not in seawater alone. By activating adenylate cyclase with L858051, an analogue of forskolin, or by activating the stimulatory form of G proteins (Gs) with cholera toxin, CSCCs are induced to sensitize CSMs and to tune VSMs to the lower frequencies of 5, 15, 30, and 40 Hz. Caged GTP-gamma S also sensitizes CSMs but tunes VSMs to 5, 15, 30, 40, 55, 65, and 75 Hz, suggesting that VSM tuning may be regulated both by Gs and inhibitory G-proteins. Together, these results implicate cAMP as the second messenger for activated supporting cell chemoreceptors involved in sensitizing the CSMs and tuning the VSMs to lower frequencies.

Control of Cnida Discharge: III. Spirocysts are Regulated by Three Classes of Chemoreceptors.

Spirocysts are two to three times more abundant than nematocysts in the feeding tentacles of acontiate sea anemones. Despite their prevalence, little experimental work has been done on the discharge of spirocysts because of the difficulty in detecting and counting them after they have discharged. To circumvent this problem, we have developed a simple, reliable, enzyme-linked lectin sorbent assay (ELLSA) for quantifying discharged spirocysts. With this method, we have shown that the discharge of spirocysts, like that of mastigophore nematocysts, is chemosensitized in a dose-dependent manner by three classes of low molecular weight substances, typified by N-acetylneuraminic acid (NANA), glycine, and certain heterocyclic amino compounds, such as proline and histamine. We also show that spirocysts exhibit considerable agonist-specific variation in the dose-responses of discharge, suggesting the existence of multiple populations of spirocyst-bearing cnidocyte/supporting cell complexes (CSCCs). Our findings call into question commonly held views regarding the respective roles of spirocysts and mastigophore nematocysts in the retention of captured prey.

Cnidocyte mechanoreceptors are tuned to the movements of swimming prey by chemoreceptors.

Cnidocytes, the stinging cells of cnidarians, discharge nematocysts in response to physical contact accompanied by the stimulation of specific chemoreceptors. Cnidocytes in fishing tentacles of a sea anemone are now found to discharge nematocysts preferentially into targets vibrating at 30, 55, and 65 to 75 hertz. Moreover, in the presence of submicromolar concentrations of known chemosensitizers, such as N-acetylated sugars and mucin, these optima shift to 5, 15, 30, and 40 hertz, frequencies that correspond to the movements of swimming prey. Hence, chemoreceptors for these substances tune cnidocyte mechanoreceptors to frequencies that match the movements of the prey.

Cnidocytes and adjacent supporting cells form receptor-effector complexes in anemone tentacles.

Cnidocytes, the stinging cells of enidarians, discharge enidae (intracellular capsules containing eversible tubules) in response to physical contact combined with the stimulation of specific chemoreceptors. These receptors, occurring in at least two classes, bind N-acetylated sugars and certain amino-compounds, respectively (Thorington and Hessinger, 1988). Colloidal gold coated with bovine submaxillary mucin (mucin-gold) binds exclusively at the surface of the supporting cells which surround enidocytes (Watson and Hessinger, 1986). We now find that mucin-gold sensitizes enidocytes to discharge nematocysts in a dose-dependent manner. In addition, we find that the number of mucin-gold particles appearing at the surface of supporting cells changes over time, and that such changes correlate with the time-course of enidocyte responsiveness. Thus, the discharge of nematocysts by enidocytes may be regulated by the number of receptor-ligand complexes at the surface of the adjacent supporting cells. We conclude that enidocytes and supporting cells constitute receptor-effector complexes. Subsequent to binding at the cell surface, mucin-gold is endocytosed (Watson and Hessinger, 1987a). Multivesicular bodies seem to dispose of the endocytosed mucin-gold at the cell surface rather than via lysosomes. This novel route appears to be the major pathway by which endocytosed mucingold is removed from supporting cells.

Receptor-mediated endocytosis of a chemoreceptor involved in triggering the discharge of cnidae in a sea anemone tentacle.

Collodial gold coated with the glycoprotein, bovine submaxillary mucin (BSM-gold), was used to localize chemoreceptors known to be involved in triggering the discharge of cnidae in sea anemones. BSM-gold binds exclusively at the apical surface of the supporting cell, the cell adjacent to the cnidocyte (Watson and Hessinger, 1986). Subsequent to binding, BSM-gold is internalized into endosomes and then translocated to multivesicular bodies (MVBs) and lysosomes. At cold temperature (4 degrees C), BSM-gold appears in endosomes near the surface of the cell but not in endosomes located more medially in the cell, nor in MVBs or lysosomes. The kinetics and sequence of intracellular translocation of BSM-gold were studied by fixing animals at various intervals following incubation in BSM-gold. Unlike that for supporting cells adjacent to non-cnidocytes, the amount of gold at the surface of supporting cells adjacent to penetrant cnidocytes does not seem to change despite considerable internalization of the mucin-probe. Apparently, free receptors replace receptor-ligand complexes in a one-for-one fashion in these cells.

Effect of Portuguese man-of-war venom on isolated vascular segments.

The purpose of this study was to characterize the mode of action of nematocyst venom from the Portuguese man-of-war (Physalia physalis) on isolated rabbit arterial ring segments, and to see if these in vitro effects were similar to those observed in the intact skeletal muscle vasculature of the dog (see Loredo et al., J. Pharmacol. Exp. Ther. 232: 301-304, 1985). The venom (0.021-4.28 micrograms/ml) produced dose-dependent relaxations of norepinephrine precontracted arterial segments from various vascular beds. Venom-induced relaxations were blocked by sodium meclofenamate (10-20 micrograms/ml), but not by atropine (6 micrograms/ml), propranolol (4-12 micrograms/ml) or quinacrine (2-4 X 10(-5) M). These results were similar to those observed in the intact skeletal muscle vascular bed of the dog and further implicate the stimulation of endogenous prostaglandin synthesis as the mechanism by which P. physalis venom dilates vasculature.

Vascular effects of Physalia physalis venom in the skeletal muscle of the dog.

The purpose of this study was to characterize the mode of action of the venom from Portuguese man-of-war (Physalia physalis) on the skeletal muscle vascular bed of dogs anesthetized with sodium pentobarbital (35 mg/kg). Venous outflow and the blood pressure difference across the hind-limb vascular bed were used to calculate changes in resistance due to close arterial injections of the venom (0.001-1 microgram/kg). The venom consistently produced dilations that were blocked by sodium meclofenamate (5 mg/kg). Atropine (1 mg/kg), propranolol, phentolamine, cimetidine (each 2 mg/kg) or tripelennamine (2.5 mg/kg) had no significant blocking effect. The venom also produced transient dilations on vascular beds predilated with histamine or prostaglandin E1, or preconstricted with norepinephrine (each 0.01 microgram/kg) or by sympathetic stimulation (12 V, 20 Hz). These data suggest that Physalia venom dilates skeletal muscle vasculature primarily by stimulation of endogenous prostaglandin synthesis.

Isolation and partial characterization of a hemolytic and toxic protein from the nematocyst venom of the Portuguese Man-of-War, Physalia physalis.

Nematocysts isolated from the stinging tentacles of the Atlantic Portuguese Man-of-War (Physalia physalis) possess a potent venom composed of several proteins. A hemolytic protein lethal to mice has been isolated from this nematocyst venom. This protein, physalitoxin, appears to be responsible for both the venom's hemolytic and lethal activities. The hemolysin has a molecular weight of approx. 240 000, a sedimentation coefficient of 7.8 S, and is rod-like in shape with a calculated axial ratio of about 1 : 10. It appears to be composed of three subunits of unequal size, each of which is glycosylated. Two of these subunits seem to have pKi values near 8.2 and the third near 5.5. Physalitoxin comprises about 28% of the total nematocyst venom protein. It is 10.6% carbohydrate by weight and represents the major glycoprotein of the venom. Physalitoxin is inactivated by concanavalin A and this inactivation can be blocked with alpha-methyl-mannoside. The inactivation by concanavalin A is temperature-dependent about 12 degrees C and the hemolytic activity of untreated venom is temperature-dependent below 12 degrees C. Physalitoxin is the first hemolytic toxin from a cnidarian to be purified directly from isolated nematocysts.

Cellular basis for tentacle adherence in the Portuguese man-of-war (Physalia physalis).

The fishing tentacles of Physalia physalis (Portuguese man-of-war) adhere to prey and human victims by the penetration of a barbed tubule connected to an intracellular nematocyst. The nematocyst is surrounded by a fibrillar system of microtubules and microfilaments that terminate in hemidesmosomal processes which anchor the nematocyst to the acellular mesoglea of the tentacle.