The zinc(II) ion has recently been implicated in a number of novel functions and pathologies in loci as diverse as the brain, retina, small intestine, prostate, heart, pancreas and immune system. Zinc ions are a required nutrient but elevated concentrations are known to kill cells in vitro. Paradoxical observations regarding zinc's effects have appeared frequently in the literature, and often their physiological relevance is unclear. We found that for PC-12, HeLa and HT-29 cell lines as well as primary cultures of cardiac myocytes and neurons in vitro in differing media, approximately 5 nmol/ L free zinc (pZn = 8.3, where pZn is defined as − log 10 [free Zn 2+ ]) produced apparently healthy cells, but 20-fold higher or (in one case) lower concentrations were usually harmful as judged by multiple criteria. These results indicate that (1) the free zinc ion levels of media should be controlled with a metal ion buffer; (2) adding zinc or strong zinc ligands to an insufficiently buffered medium may lead to unpredictably low or high free zinc levels that are often harmful to cells; and (3) it is generally desirable to measure free zinc ion levels due to the presence of contaminating zinc in many biochemicals and unknown buffering capacity of many media.

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Zinc is a structural component of many regulatory molecules including transcription factors and signaling molecules. We report that two alternate signaling pathways of protein kinase C (PKC) activation involving either the lipid second messengers (diacylglycerol and its mimetics, the phorbol esters) or reactive oxygen converge at the zinc finger of the regulatory domain. They all trigger the release of zinc ions. An increase in intracellular free Zn 2؉ was observed by confocal microscopy in intact cells treated with phorbol ester or by mild oxidation. The source of liberated Zn 2؉ was traced to PKC and particularly the zinc finger domains. The activated form of native PKC␣ contained significantly less Zn 2؉ than the resting form. Furthermore, purified recombinant PKC protein fragments shed stoichiometric amounts of Zn 2؉ upon reaction with diacylglycerol, phorbol ester, or reactive oxygen in vitro. Our results offer new insight into the regulation of PKC. Far from cementing rigid structures, zinc actually is the linchpin that orchestrates dynamic changes in response to specific signals, allowing kinase activity to be turned on or off.Zinc is essential to the structure of numerous signaling proteins. Transcription factors and signaling molecules share zinc finger structures as a common motif, although they differ in composition and function (1). The C2H2 zinc finger is believed to provide rigidity to transcription factors for proper DNA binding capacity. By comparison, the structure/function relationship of the components in the C3H1 zinc fingers of signaling molecules is not fully understood. We report here that Zn 2ϩ ions of the cysteine-rich domain play a dynamic role in the function of protein kinase C.Protein kinase C (PKC) 1 isoforms function as central signal amplifiers. They are engaged by two alternate pathways. Most isoforms with the exception of atypical ones (2) are activated by the second messenger, diacylglycerol (3, 4), or its mimetics, the phorbol esters (5). Independent of this classic pathway, PKC is also controlled by a redox mechanism where oxidation converts the protein to the catalytically competent form (6 -8), whereas reduction reverses this process (9). Cofactors are required for enzyme regulation by both pathways. Calcium and phosphatidylserine are important for amplification of the diacylglycerol signal, whereas vitamin A, as we have shown recently, is needed for efficient redox activation of several PKC isoforms (9). The lipid binding sites, accommodating diacylglycerol or phorbol ester on one hand (10, 11) and retinol on the other (12) are located at the non-overlapping regions within the twin cysteine-rich structures (referred to as Cys domains) of the regulatory domains. These 50 amino acid-long highly homologous stretches contain 6 conserved cysteine and 2 conserved histidine residues, tetrahedrically coordinated by two Zn 2ϩ ions into a composite zinc finger (1,(13)(14)(15). We show that the diacylglycerol phorbol ester pathway and the alternate redox pathway converge at this zinc...

The recent discovery of zinc signals and their essential role in the redox signaling network implies that zinc homeostasis and the function of zinc-containing proteins are probably altered as a result of oxidative stress, suggesting new targets for pharmacological intervention. We hypothesized that the level of intracellular labile zinc is changed in hearts subjected to ischemia/reperfusion (I/R) and investigated whether the maintenance of myocardial zinc status protected heart functions. Using fluorescent imaging, we demonstrated decreased levels of labile zinc in the I/R hearts. Phorbol 12-myristate 13-acetate, a known trigger of zinc release, liberated zinc ions in control hearts but failed to produce any increase in zinc levels in the I/R rat hearts. Adding the zinc ionophore pyrithione at reperfusion improved myocardial recovery up to 100% and reduced the incidence of arrhythmias more than 2-fold. This effect was dose-dependent, and high concentrations of zinc were toxic. Adding membrane-impermeable zinc chloride was ineffective. Hearts from rats receiving zinc pyrithione supplements in their diet fully recovered from I/R. The recovery was associated with the prevention of degradation of the two protein kinase C isoforms, ␦ and , during I/R. In conclusion, our results suggest a protective role of intracellular zinc in myocardial recovery from oxidative stress imposed by I/R. The data support the potential clinical use of zinc ionophores in the settings of acute redox stress in the heart.

The identification of the protein that exerts the function of Cl-/HCO3- exchange is still unresolved in cardiac tissue. We have addressed this issue by using a multiple technical approach. Western blotting analysis with an antibody raised against human erythroid whole band 3 protein, the so-called protein that mediates the Cl-/HCO3- exchange in erythrocytes, showed that adult cardiomyocytes expressed two proteins immunologically related to the erythroid band 3. These proteins migrated in SDS-polyacrylamide gel electrophoresis with apparent molecular masses of 80 and 120 kDa. They were specifically found in the membrane but not in the cytosolic or the myofibril fractions of adult cardiomyocytes. Confocal microscopy further indicated that the immunostained proteins were mainly located at the sarcolemma and along T-tubules, typical membrane structures of adult cardiomyocytes. Using an antibody raised against a cardiac amino-terminal domain of rat AE3, we found that the 120-kDa protein is the translation product of the AE3 gene specifically expressed in heart and brain. Using an antiserum raised against a specific domain of mouse erythroid band 3 (AE1), which is not shared by AE3, we showed that the 80-kDa protein is likely to be a truncated translation product of the AE1 gene. Microinjection of the anti-human erythroid whole band 3 antibody into single isolated cardiac cells significantly inhibited the Cl-/HCO3- exchange activity. Furthermore, the anti-AE1 antibody strongly decreased the efficiency of 4,4'-diisothiocyanatostilbene-2,2'-disulfonate to inhibit the ionic exchange. We thus suggest that the 80-kDa or both the 80- and the 120-kDa proteins immunologically related to the erythroid band 3 protein perform the anionic exchange in rat cardiomyocytes.