Biomedical subjects

J B Suszkiw

Publications and source records attributed to J B Suszkiw.

At least 19 recordsLinked to original sources

PubMed2000

Upregulation of tyrosine hydroxylase and downregulation of choline acetyltransferase in lead-exposed PC12 cells: the role of PKC activation.

The effects of lead (Pb) on the expression of tyrosine hydroxylase (TH) and choline acetyltransferase (ChAT) were compared in relation to Pb-activation of cPKC in the PC12 cells. Exposure to 0.53 microM Pb (0.1094 ppm) increased TH but reduced ChAT activity and mRNA levels. The increase of TH activity was detectable as early as 0.5 h of exposure, reached a maximum 150% of control after 2 h, and then diminished to a steady state 135% of control between 12 and 48 h of exposure. The decrease of ChAT activity was first detectable after 2 h of Pb exposure, reached a 45% reduction after 6 h, and remained stable thereafter through 48 h of exposure. PKC activity increased 200% after 2 h and then reverted to control levels by 48 h of Pb exposure. The increase of TH activity after 2 h but not 48 h of exposure exceeded that of its mRNA. PKC inhibitor Rö32-0342 suppressed TH activity increase after 2 h of Pb exposure by 80% without affecting TH mRNA. The decrease of ChAT activity correlated with the reductions in steady-state ChAT mRNA levels at 2 and 48 h of Pb exposure and Rö32-0342 had no effect on the Pb-induced decrease of either ChAT activity or mRNA. These results demonstrate that Pb alters TH and ChAT expression in PC12 cells in a reciprocal manner, i.e., upregulates the former but downregulates the latter. PKC is not involved in Pb-induced downregulation of ChAT but does mediate the early phase of Pb-induced augmentation of TH activity, presumably through postranslational modification (phosphorylation) of the enzyme. However, this effect is short-lived due to downregulation of PKC in the course of prolonged (48 h) Pb exposure. It is concluded that, in the course of prolonged exposure, both upregulation of TH and downregulation of ChAT reflect primarily the effects of Pb at the level of gene expression through mechanisms that are not related to Pb activation of PKC.

Animals↗

PubMed2000

Developmental stage-dependent protective effect of NGF against lead cholinotoxicity in the rat septum.

The ability of nerve growth factor (NGF) to ameliorate developmental cholinotoxicity of inorganic lead (Pb) for the septal neurons was investigated by making intracerebroventricular injections of single doses of 30 microg 2.5S NGF into maternally lead-exposed suckling rats on postnatal days P2, P4, P11, or P18. Administration of NGF on P4 or later induced septal choline acetyltransferase (ChAT) activity to the same relative extent in both Pb-exposed as in control rats but failed to reverse the net reductions of ChAT activity induced by Pb. In contrast, injection of NGF at P2 completely restored ChAT activity in Pb-exposed pups to control levels by preventing the loss of ChAT-immunoreactive cells in the septum. It is concluded that although NGF retains the capacity to upregulate ChAT throughout the period of Pb exposure, it protects against the Pb-induced loss of septal cholinergic neurons only when applied within the critical period of Pb-vulnerability between postnatal days 2 and 4.

Acetylcholine↗

PubMed1999

Analysis of differential effects of Pb2+ on protein kinase C isozymes.

Protein kinase C has been implicated as a cellular target for Pb2+ toxicity. We have previously proposed that Pb2+ modulates PKC activity by interacting with multiple sites within the enzyme. In order to further characterize the Pb-PKC interactions we compared the effects of Pb2+ on the CA-dependent and -independent protein kinase C isozymes using recombinant human PKC-alpha, PKC-epsilon, and PKC-zeta as well as the catalytic fragment of bovine brain protein kinase C, the PKC-M. The results demonstrate that, whereas at pM concentrations Pb2+ activates PKC-alpha half maximally (KAct approximately 2 pM), it has no effect on PKC-epsilon, PKC-zeta, or PKC-M activities. The activation of PKC-alpha by Pb2+ is additive with Ca2+ in a manner indicating interaction with half of the calcium activation sites. In the micromolar range of concentrations, Pb2+ inhibits all PKCs with estimated K0.5 of 1.0, 2.3, 28, and 93 microM for PKC-M, PKC-alpha, PKC-epsilon, and PKC-zeta, respectively. Examination of Pb2+ effects on PKC-M kinetics indicates a mixed type inhibition with respect to ATP and noncompetitive inhibition with respect to histone. Taken together with the results of our previous study (Tomsig and Suszkiw, J. Neurochem. 64, 2667-2673, 1995) and the evidence for the existence of two Ca2+ coordination sites Ca1 and Ca2 within the C2 domain (Shao et al., Science [Washington, D.C.] 273, 248-251, 1996), the results of the current study provide further support for a multisite Pb-PKC interaction scheme wherein lead (1) partially activates the enzyme through pM-affinity interactions with the Ca1 site and inhibits the divalent cation-dependent activity through nM-affinity interactions with Ca2 site in the C2 domain and (2) inhibits the constitutive kinase activity through microM-affinity interactions with the catalytic domain. The concentration dependence of the differential effects of Pb2+ on the calcium-dependent and -independent PKCs underscores the importance of the C2 motif as a high affinity molecular target for Pb2+.

Amino Acid Sequence↗

PubMed1998

Symposium overview: chemical modulation of neuroreceptors and channels via intracellular components.

Whereas the roles of G proteins and protein kinases in various neuroreceptors and ion channels have been studied extensively, their roles in the actions of drugs and toxicants on these receptors and channels remain to be elucidated. Almost all drugs and toxicants exert multiple actions on multiple target sites, and there is no reason to assume that a chemical modulates a receptor/channel via a single mechanism. In fact, experimental evidence is slowly but steadily being accumulated to indicate that certain drugs and toxicants modulate neuroreceptor/channel functions through interactions with intracellular components such as G proteins and protein kinases. Multiple actions of a toxicant on various receptors/channels may be explained on the basis of its interaction with the G protein/kinase system that is a common denominator of the target sites. This is a virgin field that promises a quantum leap in the coming years. Each presentation and discussion will focus on expected future developments and potential significance in the field of neurotoxicology.

Animals↗

PubMed1997

Developmental cholinotoxicity of lead: loss of septal cholinergic neurons and long-term changes in cholinergic innervation of the hippocampus in perinatally lead-exposed rats.

The effects of perinatal lead exposure on choline acetyltransferase-immunoreactive (ChAT-IR) cell counts in the medial septum and AChE-positive fiber counts in the hippocampus were examined in relation to changes in cholinergic markers in the septohippocampal pathway of the rat. Maternal exposure to 0.2% lead acetate in drinking water from gestational day 16 through weaning at post-natal day 21 (P21) induced in the offspring a 30% reduction in septal ChAT activity and a 20% reduction in ChAT-IR cell profile counts in the medial septum/vertical diagonal band (MS/vDB). These changes were seen as early as P7, persisted through 2 months post-exposure (P81), and were followed by recovery of ChAT activity but not the ChAT-IR cell numbers, at 3 months post-exposure (P112). The loss of ChAT activity and ChAT-IR neurons in the septum was temporally associated with a reduction of ChAT activity (30%), hemicholinium-3 (HC-3) binding (40%), and AChE-positive fiber counts (13-15%) in the hippocampus. The hippocampal ChAT activity and AChE-positive fiber counts returned to control levels by P112 whereas HC-3 binding was restored to normal levels by P200. These results indicate that perinatal, low-level lead exposure induces loss of septohippocampal cholinergic projection neurons in neonate animals, resulting in a deficit in hippocampal cholinergic innervation that persists into young adulthood. The disruption of cholinergic septohippocampal system may be an important factor in lasting cognitive impairments associated with early Pb exposure.

Acetylcholinesterase↗

PubMed1997

Reduction of vesicular acetylcholine transporter mRNA in the rat septum following lead exposure.

We have previously observed that maternal exposure to lead (Pb) results in a reduction of levels of mRNA coding for cholineacetyltransferase (ChAT) in the septum of developing rat without affecting the dams. Here we report that Pb similarly affects the expression of vesicular acetylcholine transporter (VAChT) mRNA in the rat septum. In close agreement with the time course of ChAT mRNA expression, septal VAChT mRNA levels increased from 30% at postnatal day 7 to 78% and 100% of adult levels at days 14 and 21, respectively. Maternal exposure to 0.2% lead acetate in drinking water from gestational day 16 resulted in an approximately 30% reduction of VAChT in 7 and 21-day-old rat pups without affecting VAChT mRNA levels in the dams. These results indicate a developmental stage-dependent interference by Pb with ChAT/VAChT gene expression in the rat septum.

Acetylcholine↗

PubMed1997

Reduction of choline acetyltransferase mRNA in the septum of developing rat exposed to inorganic lead.

We previously reported that perinatal exposure to low levels of lead (Pb) results in a pronounced reduction of choline acetyltransferase (ChAT) activity in the septohippocampal pathway of the rat. In the present study we employed a ChAT specific 43-base oligonucleotide hybridization probe to compare the developmental expression of ChAT mRNA and ChAT activity in the septum of normal and Pb-exposed rats. Rats were exposed to Pb via maternal administration of 0.2% solution of Pb acetate in drinking water from one week before birth through postnatal day 21 (PN21). In normal animals, the developmental changes in the ChAT mRNA and activity levels were temporally correlated and approached adult levels by PN21. In the Pb-exposed pups, ChAT mRNA and activity levels were reduced respectively by 69% and 50% at PN7, 47% and 33% at PN14, and 47% and 22% at PN21. In contrast, Pb exposure had no significant effect on either ChAT mRNA or ChAT activity in the dams. These results indicate that cholinotoxicity of Pb in the septohippocampal pathway of the rat involves developmental stage-dependent disruption of ChAT gene expression.

Animals↗

PubMed1996

Developmental age-dependent upregulation of choline acetyltransferase and vesicular acetylcholine transporter mRNA expression in neonatal rat septum by nerve growth factor.

We examined the effect of intraventricular injection of nerve growth factor (NGF) on the choline acetyltransferase (ChAT) and vesicular acetylcholine transporter (VAChT) mRNA expression in the septa of neonatal rats. Rat pups were injected with 2.5 S NGF or cytochrome-c (control) on postnatal days (PN) 4 and 18, and sacrificed 3 days after injections for analysis of ChAT and VAChT mRNA levels by dot-blot hybridization of total septal RNA. In the NGF-treated pups, the ChAT and VAChT mRNA levels were elevated 3- and 2-fold, respectively, at PN7, and 1.8- and 1.3-fold at PN21. These results indicate that (1) NGF upregulates the expression of both ChAT and VAChT genes, (2) NGF has a greater effect on the expression of ChAT mRNA than VAChT mRNA, and (3) the effect of exogenous NGF on the expression of both genes diminishes with developmental age.

Age Factors↗

PubMed1996

Cholinergic denervation-like changes in rat hippocampus following developmental lead exposure.

We investigated the effects of developmental lead exposure from embryonic day 16 (E16) through postnatal day 28 (PN28), on cholinergic and catecholaminergic markers in the septohippocampal pathway in rats through fourth month of age. Lead exposure resulted in a persistent 30-40% reduction of [3H]hemicholinium-3 ([3H]HC-3) binding in the hippocampus through PN120, and 20-30% reduction of septal and hippocampal choline acetyltransferase (ChAT) activity which persisted through PN84 but returned to control levels in both septum and hippocampus at PN112. The muscarinic ligand [3H]quinuclidinyl benzylate ([3H]QNB) binding was reduced in the septum at PN28 but did not differ significantly from controls at PN56-PN112. Neither short- nor long-term effects of Pb exposure on [3H]QNB binding were seen in the hippocampus. Similar to the effects of fimbria-fornix transection, Pb exposure resulted in a long-term 50-90% increase of tyrosine hydroxylase(TH) activity in the hippocampus, although neither treatment affected TH activity in the septum. The lead-induced increase in hippocampal TH was significantly attenuated by superior cervical ganglionectomy. It is concluded that the effects of perinatal lead exposure resemble in several respects those seen following surgical disruption of the septohippocampal pathway in adult animals. The denervation-like effects in the hippocampus may be an important factor in long-term learning and cognitive impairments following developmental exposure to low-levels of lead.

Aging↗

PubMed1996

Metal selectivity of exocytosis in alpha-toxin-permeabilized bovine chromaffin cells.

Metal selectivity of exocytosis was analyzed by comparing the effects of polyvalent metal cations Ca2+, Ba2+, Sr2+, Pb2+, La3+, Cd2+, Co2+, Tb3+, Mn2+, and Zn2+ on the release of norepinephrine (NE) from staphylococcal alpha-toxin-permeabilized bovine chromaffin cells. Pb2+, La3+, Cd2+, Sr2+, and Ba2+ activated NE secretion accompanied by the release of intragranular dopamine beta-hydroxylase but not cytosolic lactate dehydrogenase, indicating the activation of the mechanism of exocytosis. The release triggered by saturating concentrations of Pb2+, La3+, Cd2+, and Sr2+ was nonadditive with Ca2+, indicating a common site of action. In contrast, the Ba2(+)-evoked NE release was additive with Ca2+ and the Ca2+ agonists Pb2+, La3+, Cd2+, and Sr2+, suggesting that Ba2+ activates secretion at a site distinct from the Ca2+ receptor. In distinction to the NE release evoked by Pb2+, La3+, Cd2+, and Ba2+, the Sr(2+)-evoked NE release was associated with a significant elevation of Ca2+ concentration in the medium and abolished by Ca2+ chelation. This indicates that the secretagogue effect of Sr2+ was indirect and secondary to the displacement of bound Ca2+, Co2+ and Mn2+ inhibited the NE release evoked by Ca2+, Sr2+, Pb2+, La3+, and Cd2+ but had no effect on the Ba(2+)-dependent secretion. Tb3+ and Zn2+ were without effect on exocytosis.

Adrenal Medulla↗

PubMed1995

Reduced densities of sodium-dependent [3H] hemicholinium-3 binding sites in hippocampus of developmental rats following perinatal low-level lead exposure.

We investigated the effect of perinatal, low-level lead exposure on [3H]hemicholinium-3 (HC-3) binding in the hippocampus of postnatal rat. Rat pups were maternally lead-exposed from gestational day 16 through postnatal day 28 (PN28). In control animals, the [3H]HC-3 binding sites increased from 7 fmol/mg protein at postnatal day 1 (PN1) to 14 and 35 fmol/mg protein at PN7 and PN14, respectively, and reached adult values of 50 fmol/mg protein, at PN21 and PN28. In lead-exposed litters, the [3H]HC-3 binding was reduced by 30-40% throughout the early postnatal development and remained 40% below control values in PN60 animals, one month after termination of lead exposure. The Pb-induced reduction in HC-3 binding was associated with a similar decrease in ChAT activity and was comparable to the effect of localized lesion of medial septum. Septal cell counts in the lead-exposed PN21 rats indicated a 22% reduction in the number of ChAT-immunoreactive cells in the medial septum/vertical diagonal band (MS/vDB) complex although cell numbers in the horizontal limb of the diagonal band (hDB) were not altered. These results suggest that perinatal, low-level lead exposure results in a reduced density of cholinergic nerve terminals in the hippocampus, either due to impaired development or degeneration of the cholinergic projection neurons in the MS/vDB complex.

Aging↗

PubMed1995

Multisite interactions between Pb2+ and protein kinase C and its role in norepinephrine release from bovine adrenal chromaffin cells.

We investigated the interaction between Pb2+ and protein kinase C (PKC) in the Pb(2+)-induced release of norepinephrine (NE) from permeabilized adrenal chromaffin cells. Our analysis of endogenous PKC activity in permeabilized cells suggests that Pb2+ interacts with the adrenal enzyme at multiple sites. Pb2+ activates the enzyme through high-affinity (KA(Pb) = 2.4 x 10(-12) M) interactions and inhibits the enzyme by competitive and noncompetitive interactions with nanomolar-(Ki = 7.1 x 10(-9) M) and micromolar-(Ki = 2.8 x 10(-7) M) affinity sites, respectively. Activation of PKC by 12-O-tetradecanoyl-phorbol 13-acetate (TPA) in Ca(2+)-deficient, Pb(2+)-containing medium, enhances the Pb(2+)-induced NE release from permeabilized chromaffin cells by lowering the concentration of Pb2+ required for half-maximal activation of the secretory response from 7.5 x 10(-10) to 5.7 x 10(-11) M. The PKC inhibitors staurosporine and pseudosubstrate PKC (19-36) abolish the effect of TPA without affecting the Pb(2+)-induced secretion in the absence of TPA. These results indicate that (a) Pb2+ is a partial agonist of PKC, capable of both activating and inhibiting the enzyme and (b) synergistic activation of PKC by TPA and Pb2+ results in increased sensitivity of exocytosis to Pb2+ but is not obligatory for Pb(2+)-triggered secretion.

Adrenal Glands↗

PubMed1995

Extracellular inhibition and intracellular enhancement of Ca2+ currents by Pb2+ in bovine adrenal chromaffin cells.

1. Effects of highly neurotoxic, inorganic lead ions (Pb2+) on voltage-dependent calcium channels were investigated with the use of the whole cell patch-clamp technique in bovine adrenal chromaffin cells maintained in short-term primary culture (1-5 days). 2. Extracellularly applied Pb2+ induced a concentration-dependent, reversible inhibition of Ca2+ currents, with an estimated IC50 approximately equal to 3.0 x 10(-7) M free Pb2+. 3. Elevation of the intracellular free Ca2+ concentration above 10(-8) M dose-dependently reduced the amplitude of the initial Ca2+ current and increased the exponential rate of current rundown. 4. Intracellularly applied Pb2+ prevented the Ca(2+)-dependent reduction of the initial Ca2+ current amplitude and altered the current rundown kinetics from exponential to linear. The effect was dose dependent and saturable, with an estimated EC50 approximately equal to 2.0 x 10(-10) M free Pb2+. 5. These results indicate that in contrast to extracellular blockade, intracellular Pb2+ promotes Ca2+ currents by attenuating the Ca(2+)-dependent, steady-state inactivation of calcium channels. This provides a novel mechanism through which Pb2+ may disrupt calcium signaling in chronically lead-exposed cells.

Adrenal Glands↗

PubMed1994

Pb2+ activates potassium currents in bovine adrenal chromaffin cells.

Inorganic lead (Pb2+) is a potent environmental toxin which adversely affects several aspects of neuronal and secretory cell function. In this report, we provide evidence that at subnanomolar concentrations, Pb2+ activates the outward K+ currents in bovine adrenal chromaffin cells. Whole-cell patch clamp combined with intracellular perfusion was employed to monitor outward K+ currents in bovine chromaffin cells before and after intracellular application of EGTA-Pb buffers. Intracellular Pb2+ > or = 10(-10) M enhanced the K(+)-currents in a concentration dependent manner, with apparent K0.5 approximately equal to 5 x 10(-10) M. Extracellular application of 40 nM Charybdotoxin (ChTX) blocked the Pb(2+)-dependent component of outward currents, suggesting that Pb2+ activates the large conductance Ca(2+)-dependent K+ channels.

Adrenal Glands↗

PubMed1994

Perinatal low-level lead exposure and the septo-hippocampal cholinergic system: selective reduction of muscarinic receptors and cholineacetyltransferase in the rat septum.

We investigated the effects of low-level lead exposure on the postnatal development of cholinergic muscarinic receptors (mAChR) and a cholinergic marker enzyme cholineacetyltransferase (ChAT) activity in the rat septum and hippocampus. Rat pups were maternally lead-exposed by giving 0.2% lead acetate in drinking water to dams from one week before parturition (gestational day 16) through weaning at postnatal day 28. The lead-exposed litters had blood Pb in the range 20 micrograms/dl and tissue Pb < 0.2 micrograms/g in both the septum and hippocampus. Associated with this level of lead exposure there was a significant 30-40% reduction in the ChAT activity in the septa and hippocampi of PN7 through PN28 animals. In contrast, the levels of glutamic acid decarboxylase (GAD) activity, a GABAergic neuron marker enzyme, were not altered in either brain region. Associated with the selective reduction of ChAT activity there was a parallel 30-40% reduction of the [3H]quinuclidinyl benzilate, [3H]AF-DX 384, and [3H]pirenzepine binding in the septum, however muscarinic ligand binding in the hippocampus of lead exposed animals was not affected. These results indicate preferential vulnerability of septal cholinergic neurons to adverse effects of low-level Pb exposure and suggest that impaired expression of muscarinic receptors and disruption of muscarinic transmission in the septum may be an important factor in cognitive and learning deficits associated with developmental low-level lead exposure.

Aging↗

PubMed1993

Intracellular mechanism of Pb(2+)-induced norepinephrine release from bovine chromaffin cells.

The intracellular mechanism of Pb(2+)-induced release of norepinephrine (NE) was investigated in comparison with Ca2+ in bovine chromaffin cells permeabilized with staphylococcal alpha-toxin. Pb2+ activated NE release at considerably lower concentrations [concentration of free metal giving half maximal metal-dependent release (K0.5) 4.6 nM] than Ca2+ (K0.5 2.4 microM). The release of NE was associated with the release of dopamine-beta-hydroxylase but not lactate dehydrogenase. The maximal secretory responses produced by Pb2+ and Ca2+ were similar and nonadditive. Pb(2+)- and Ca(2+)-dependent releases showed a similar requirement for MgATP and were equally enhanced by protein kinase C activator 12-O-tetradecanoylphorbol 13-acetate (TPA) but not by kinase A activator 8-bromoadenosine 3',5'-cyclic monophosphate free base. The protein kinase C inhibitor staurosporine blocked the TPA-stimulated component of secretion but had no effect on the NE release in the absence of TPA. Calmidazolium, an inhibitor of calmodulin, inhibited the secretion evoked by both metals to similar extent. Agents interacting with microtubules (colchicine and vinblastine) or microfilaments (cytochalasin B and phalloidin) had no effect on secretion induced by either metal cation. These observations indicate that both Pb2+ and Ca2+ act at a common site and activate the exocytotic release of NE by an analogous mechanism.

8-Bromo Cyclic Adenosine Monophosphate↗

PubMed1992

Electrophysiological properties of early postnatal rat septal neurons in short-term culture.

Electrophysiological properties of septal neurons dissociated from PN1-PN7 rats were examined between 1 and 5 days in vitro (DIV) with whole-cell patch-clamp recording. The neurons had RMPs in the range -40 to -80 mV, resistances 0.5-1.5 G omega, and 50-90 mV action potentials. By 2-3 DIV, most neurons were spontaneously active, with some cells exhibiting rhythmic firing patterns. Depolarizations from -80 mV holding potential elicited TTX-sensitive inward Na+ currents, and transient and sustained outward K+ currents. Pharmacological dissection of the outward currents in PN6/7 neurons suggest the presence of multiple types of K+ currents (IA, IC, IK). L-type Ca2+ currents were observed in all PN6/7 neurons examined but were not always detectable in PN1/2 neurons. All PN1/2 and PN6/7 neurons were sensitive to glutamate and GABA but did not respond to ACh or NE applications. Responses to GABA were excitatory i.e., characteristic of immature neurons. Comparison of the results obtained in this study with the properties of adult neurons characterized in vivo or in brain slice preparations in vitro, suggest that septal neurons from PN1-PN7 rats are still in the process of differentiation of their mature electrical and chemosensitive membrane properties.

Adenosine Triphosphate↗

PubMed1991

Permeation of Pb2+ through calcium channels: fura-2 measurements of voltage- and dihydropyridine-sensitive Pb2+ entry in isolated bovine chromaffin cells.

Fura-2 was used to monitor Pb2+ entry into isolated bovine chromaffin cells exposed to micromolar concentrations of Pb2+ in media containing basal or high concentrations of K+. The entry of Pb2+ consists of voltage-independent and voltage-dependent (K(+)-stimulated) components. The voltage-dependent Pb2+ entry is enhanced by Ca2+ channel agonist BAY K 8644 and blocked by the channel antagonist nifedipine, suggesting the involvement of the L-type Ca2+ channels. In contrast to the transient, K(+)-depolarization-dependent increase in [Ca2+]i, the increase in [Pb2+]i is sustained over a period of several minutes, suggesting the absence of channel inactivation and/or the saturation of Pb(2+)-buffering capacity of the cell cytosol.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗