Intracellular Zn²⁺ must be tightly regulated to prevent the adverse effects of zinc deficiency or toxicity, but since it cannot pass through biological membranes unassisted, two families of zinc-transport proteins aid in this process. A total of 10 ZnT and 14 ZIP gene loci in humans allows for the fine-tuning of Zn²⁺ distribution among tissues and intracellular organelles, such as the endoplasmic reticulum (ER) or Golgi apparatus [1523242526]. ZnT and ZIP proteins are energy independent, and their production is regulated by Zn²⁺ levels [27]. The fundamental role of Zn²⁺ flux from intracellular stores can be anticipated from the ubiquity of ZIP7 among different organisms and cell types [28], since Zn²⁺ release from stores is stimulated without the addition of exogenous zinc [15]. Most of the ZIP family is predicted to have eight transmembrane domains with extracellular and intravesicular amino- and carboxyl-termini, while most ZnT transporters have six. The metal binding domain of ZnTs is represented by a common histidine-rich loop [29], and this transporter promotes zinc efflux from cells or into intracellular vesicles to reduce intracellular Zn²⁺ availability. However, ZIPs do the opposite by enabling the uptake of extracellular Zn²⁺, and possibly releasing vesicular Zn²⁺ into the cytoplasm. These transporter families exhibit tissue-specific expression and heterogeneous sensitivity to dietary zinc deficiency and excess, as well as to other physiologic stimuli via hormones and cytokines [30].

MT is a small protein (~14 kDa) and comprises a family of cysteine (cys)-rich proteins [31]. The human body synthesizes MT in large quantities, mainly in the liver, kidney, intestine, and pancreas. MTs are localized in the membrane of the Golgi apparatus. The thiol group of its cysteine residues (~30% of its amino acid residues) allows MTs to bind both physiological and xenobiotic heavy metals. The homeostasis of zinc in a system is achieved by the coordination of MT with metal-free thionein, whose production is induced by increased zinc availability. The specific knockout of MT genes in mice with multiple MT-1 genes does not exhibit major developmental defects, suggesting a minimal role in normal growth and development; however, during conditions of major infection and inflammation, MTs have been known to provide survival advantages [32].

MT gene expression is triggered by various stimuli, and the MT promoter region has specific sequences for metal response elements (MRE), glucocorticoid response elements, GC-rich boxes, basal level elements, and thyroid response elements [32]. Transcriptional activation also occurs at higher zinc levels, as observed in the gene regulatory response mediated by MRE-binding transcription factor 1 (MFT1) to copies of the MRE consensus sequence in the promoter region [33]. MT also serves as a free radical scavenger [31]. Mammalian MT-1/MT-2 isoforms are involved in zinc homeostasis and protection against heavy metal toxicity and oxidative stress. MT-3 is expressed mainly in neurons, but also in glia [34], while MT-4 is mostly present in differentiating stratified squamous epithelial cells [31].

As extracellular Zn²⁺ enters the cell, it is immediately buffered within a 'zinc muffler' via a still unidentified Zn²⁺ transporter, and is then translocated into an intracellular zinc store, such as the ER or Golgi [35]. The Zn²⁺ is then released into the cytosol from the ER through the action of ZIP7, creating a zinc wave [36]. Mitochondria are another possible site of intracellular zinc sequestration. Zn²⁺ can be released from zinc-bound MT, physiological Zn²⁺ ligands [37], cysteine, histidine, glutathione (GSH), aspartate, glutamate, and glycine, in an acidic condition such as in ischemia [3839], from ER and Golgi through ZnTs, mitochondria with unknown mechanisms [40], or acidic organelles [41]. The partial cationic character of Zn²⁺ enables it to attract anionic prooxidants, such as peroxynitrite (ONOO^-). For example, zinc is released from endothelial nitric oxide synthase (eNOS) upon exposure to peroxynitrite, which results in dimer disruption and enzyme uncoupling [42]. Maret and colleagues demonstrated that the presence of GSH enabled a higher transfer rate, and caused the release of more of the available Zn²⁺ from MT by glutathione disulfide (GSSG), and, thereafter, can facilitate Zn²⁺ removal and addition to inhibitory sites on enzymes involved in glycolysis and signal transduction [43]. A pH-dependent release of Zn²⁺ by cytosolic proteins is evident with cysteine, wherein the liberation of Zn²⁺ rapidly accelerates when the pH is decreased from 6.6 to 6.1 [37]; however, MTs are not likely to release Zn²⁺ at the same pH trend [39] and Zn²⁺ release from GSH-Zinc is less sensitive to an acidic pH (pH 6.1) [37]. At physiological pH, cytosolic proteins and ligands prevent large increases in intracellular Zn²⁺ by rapidly buffering the Zn²⁺ released from the mitochondria. When the mitochondrial matrix pH drops from 8 to 7, GSH starts to release Zn²⁺, which then leaks into the cytosol. At a pH of 7, ligands, such as MTs and cysteine, intercept the released Zn²⁺; only after the pH drops below 6.6 and cysteine loses its affinity for Zn²⁺ does the elevation of intracellular Zn²⁺ levels become evident [37].

In cardiomyocytes or other cells, the intracellular zinc concentrations are 100-fold less than those of intracellular Ca²⁺ [444]. The relatively low abundance of Zn²⁺ in cardiomyocytes suggests its availability as second messengers for gene expression [45]. The inhibition of cyclic AMP (cAMP) signaling by Zn²⁺ has been attributed to both an inhibition of adenylyl cyclase (AC), particularly the AC5 and AC6 isoforms [4647] that are present in the cardiomyocytes [48], and of the function of the Gs alpha subunit (Gαs). Zn²⁺ induces aggregates of pure tubulin, implying the activation of G proteins by tubulin dimers [49]. This possible regulation of cAMP signaling by Zn²⁺ will provide complex outcomes of cAMP and cAMP-protein kinase A signaling, which are initiated by various kinds of chemicals and hormones. Therefore, cAMP-mediated signaling may be influenced or amplified at different levels of cellular Zn²⁺.

Protein tyrosine phosphatase 1B (PTP1B) inhibits phosphoinositide 3-kinase, a key enzyme in the insulin and leptin signaling pathways, making it a negative regulator of both signaling pathways [50]. Zn²⁺ binds to the catalytic pocket of the phosphointermediate form of PTP1B at a nanomolar range [51], and works as an inhibitor of protein tyrosine phosphatases (PTPs) on account of an active site HC(X5)R signature motif and the nucleophilic attack of the phosphorus atom of the phosphate group by the sulfur ion of the cysteine thiol in the first step of the dephosphorylation reaction and/or formation of inactive dimers between thiolates of PTPs and zinc [1651]. ER-bound PTP1B is proposed to be inhibited by ZIP7-mediated Zn²⁺ release from the ER, because the co-localization of ZIP7 and PTP1B does not require a global increase in cytosolic Zn²⁺ in order to inhibit the enzyme. The inhibitory effect of Zn²⁺ on PTPs implicates zinc-induced signaling in the increase in tyrosine phosphorylation of insulin receptor (IR), insulin-like growth factor 1 receptor (IGF-1R), epidermal growth factor receptor (EGFR), or unrecognized receptor tyrosine kinases, depending on the cell type [52]. Moreover, both insulin and adiponectin directly work together with Zn²⁺, affecting their oligomerization, and hence their activities [21]. Therefore, an elevation in disturbances in intracellular Zn²⁺ status observed in metabolic disorders, such as obesity and diabetes, may directly impact leptin signaling by PTP1B inhibition [53]. In insulin-resistant rodent models, key regulators of the insulin signaling pathway, extracellular-signal-regulated kinases 1 and 2 (ERK1/2) and protein kinase B (PKB), were activated by Zn²⁺, and insulin-mimetic and anti-diabetic properties were also observed [52].

The cell's response to a perturbation in its surrounding environment heavily relies on the regulation of protein activity. Reactive disulfide bonds act as molecular switches, and proteins with closely spaced cysteines or cysteine-coordinating Zn²⁺ centers, including MT, may reveal that thiol-disulfide exchanges play a much more widespread role in protein regulation at the post-translational level than had been previously anticipated [54]. Intracellular Zn²⁺ levels can be increased by thiol-reactive oxidants. In cardiomyocytes, reactive oxygen species (ROS) are major contributors to a rapid increase in intracellular Zn²⁺ levels, due to the mobilization of Zn²⁺ from intracellular stores. Moreover, during oxidative stress, including ultraviolet A irradiation, Zn²⁺ is released from MTs, either by nitrosylation or thiol ligand oxidation [5556]. Intracellular Zn²⁺ may also contribute to oxidant-induced alterations of excitation-contraction coupling [55], changes in signal transduction, and alterations of mitochondrial function due to modification of protein structure or functions by Zn²⁺ release, or its binding to proteins [29]. These findings explain why antioxidant intervention, resulting in the prevention of both Zn²⁺ and Ca²⁺ leaks, is helpful against diabetic heart disease [4557]. Zn²⁺ plays a central role in the redox signaling network, in that when it binds to a sulfur ligand, it generates a redox-active environment, which allows conformational changes of the proteins it is attached to, activating several signaling molecules. For example, the activation of protein kinase C (PKC) could be initiated by the release of Zn²⁺ from its zinc finger regulatory domain by lipid second messengers or oxidation [58]. Another example is the Kv4.1 channel, a voltage-gated potassium channel that is a key regulator of membrane excitability in neuronal and cardiac tissues [5960]. Under physiological conditions, the native Kv4 channel complex activity is regulated by thiol-disulfide exchange reactions at the Zn²⁺ site upon exposure to reactive nitrosative or oxygen species. Intracellular nitric oxide (NO) rapidly inhibits the Kv4.1 channel in a Zn²⁺-dependent manner through a disulfide bridge across the T1-T1 interface (Cys¹¹⁰ to Cys¹³²), which may be responsible for the nitrosative/oxidative modulation of rapidly inactivating (A-type) K⁺ currents in the heart and smooth muscle, as well as in neurons [59]. Other Kv2 and Kv3 channels share the similar T1-T1 Zn²⁺ site, and thus they also can be regulated by zinc-dependent redox modulation.

Numerous proteins are targets of Zn²⁺ binding, and this direct binding of Zn²⁺ to proteins is able to directly alter the function of proteins, including ion channels. Zn²⁺ is able to induce changes of the ionic current and significantly impede the activation kinetics of most K⁺ channels through its direct binding [60]. In addition, Zn²⁺ is among the most potent inhibitors of voltage-gated proton (H⁺) and calcium channels [6162]. The abundance of potential synaptic targets and the localization of Zn²⁺ in synaptic vesicles all seem to point to a role of Zn²⁺ in synaptic transmission [63]. The entry of Zn²⁺ in cortical neurons is mediated by L- and N-type voltage-gated Ca²⁺ channels, which may be enhanced by an acidic extracellular pH [64], whereas Zn²⁺ entry into heart cells is via dihydropyridine-sensitive Ca²⁺ channels and is reliant on electrical stimulation [65]. In the dorsal root ganglion (DRG) neurons, the rise in nanomolar concentrations of intracellular Zn²⁺ can activate transient receptor potential ankyrin 1 (TRPA1), which is a sensory receptor for environmental irritants and both oxidative and thiol-reactive compounds, possibly through interaction with the intracellular cysteine and histidine residues of TRPA1, and thus potentiates the perception of discomfort [6667].

Mitochondrial enzymes are also influenced by Zn²⁺ or oxidative stress-related Zn²⁺ release, and its dysregulation can take a toll on mitochondrial metabolism [40686970]. Although there is limited information available regarding Zn²⁺-dependent mitochondrial functions, changes in mitochondrial metabolism could be introduced by the direct binding of Zn²⁺ to mitochondrial proteins or the release of Zn²⁺ from its bound proteins, due to oxidative stress or redox switches, which result in an alteration of its enzymatic activity and function [71]. In the mitochondrial matrix, GSH has a more prominent action in Zn²⁺ chelation than it does in the cytosol. The application of a sulfhydryl reactive agent, N-ethylmaleimide, leads to a major intracellular Zn²⁺ release, indicating a large amount of zinc storage in Zn²⁺-cysteine complexes [3972]. While GTP binds to MT [73], ATP also binds to mammalian MT-2 [74]. Thus, it has been proposed that released Zn²⁺ from MT may be involved in the inhibition of ADP-stimulated respiration, or the formation of an ATP-Zn²⁺ complex, which is a preferred cofactor of pyridoxal kinase and flavokinase, which will be able to influence energy metabolism [74].

Zinc can inhibit mitochondrial electron transport by binding Zn²⁺ in complexes [7576] and stimulating a burst of mitochondrial ROS production [77]. Several zinc-regulated enzymes have been identified. Fructose 1, 6-diphosphatase, glyceraldehyde 3-phosphate dehydrogenase, aldehyde dehydrogenase, and lipoamide dehydrogenase (LADH) are inhibited by nanomolar concentrations of Zn²⁺ [7178]. Inside the mitochondrial matrix, an elevated Zn²⁺ concentration interrupts the Krebs cycle and mitochondrial energy metabolism by inhibiting the production of a reduced form of nicotinamide adenine dinucleotide (NADH) production. Besides, the Zn²⁺ pool in prostate mitochondria (0.1~1 µg zinc/mg protein) is essential for inhibiting mitochondrial aconitase in the Krebs cycle, possibly through MT-mediated Zn²⁺ transfer [79], which results in the accumulation of citrate for secretion into prostatic fluid [8081]. In neurons, excessive Zn²⁺ could induce mitochondrial swelling and ROS release. However, whether or not this induced swelling also occurs in the liver remains controversial [8283].

In mitochondria-mediated apoptosis, Zn²⁺ increases the Bax-associated mitochondrial pore forming process that releases cytochrome C, activating the caspase cascade that leads to apoptosis [84]. Zn²⁺ also increases the gene expression and cellular production of Bax and the Bax/Bcl-2 ratio, which facilitates the mitochondrial Bax-associated apoptogenic effect [85]. Zn²⁺ chelation at the onset of neuronal ischemia attenuates mitochondrial cytochrome C release and downstream caspase-3 activation [86]. In contrast to brain ischemia [38], Zn²⁺ is considered as an important modulator involved in cardioprotection upon reperfusion, since several suggested cardioprotectants share zinc activation [10].

In the mammalian brain, vesicular zinc is co-localized with glutamate in a subset of glutamatergic nerve terminals throughout the forebrain. Zn²⁺ also shows anti-depression effects [92], which are associated with the action of G protein-coupled receptor 39 (GPR39), which is recognized as a Zn²⁺ receptor, and is a potent stimulator of GPR39 activity via the Gα(q), Gα(12/13), and Gα(s) pathways [9394]. Impairments of the central nervous system (CNS), such as defects in learning and memory, may be brought about in the fetus by maternal zinc deficiency, which cannot be reversed by subsequent supplementation of zinc to the diet after weaning [95]. This CNS defect may be associated with a reduction in the number of proliferating stem cells and neuronal precursor cells due to inadequate amount of available zinc [9697]. In various neuronal diseases, such as stroke, epilepsy, mechanical trauma, and Alzheimer's disease, the precise mechanism of cytotoxicity is less known, but emerging evidence suggests that excess Zn²⁺ may kill neurons through the inhibition of ATP synthesis [98]. In contrast to the previously mentioned proapoptotic role of Zn²⁺ [84], Zn²⁺ could participate in the inhibition of proapoptotic caspase-3 activation [71] and in the reduction of amyloid production in experimental neuronal cells [99], since these effects are lost in response to low levels of Zn²⁺. In zinc deficiency, neural cell apoptosis was associated with an increase in phosphorylation of p53 and its translocation into mitochondria [97].

The extracellular and intracellular levels of Zn²⁺ may be a determinant to cardiovascular health [29], and zinc deficiency related to diseases and aging may be a factor in the development of heart failure. Moreover, a potential role of zinc in diabetic cardiomyopathy has been suggested, due to its involvement in insulin signaling and diabetes development [100101]. In regards to the cardioprotective roles of Zn²⁺, four beneficial roles of Zn²⁺ were suggested: (1) protective effects from acute redox stress in cardiomyocytes; (2) prevention of inflammatory processes triggered during myocardial damage; (3) wound healing; and finally (4) maintenance of cardiac stem cells essential for cardiac healing [29].

In the heart, a large portion of intracellular Zn²⁺ accumulates in specific cellular structures, such as in or near the terminal sarcoplasmic reticulum (SR) [65]. Calsequestrin is a Zn²⁺-binding protein residing in the SR and can bind up to 200 mol of Zn²⁺ per mol [55]. Besides changes in the phosphorylation status of the L-type calcium channel (LTCC), thiol oxidation, and nitrosylation, the extracellular and intracellular levels of Zn²⁺ influence transmembrane Ca²⁺ movements via the LTCC. Moreover, intracellular Zn²⁺ levels could inhibit AC at even low concentrations (<1 nM), which strikingly reduces β-adrenergic stimulation [47]. Moreover, besides being cell permeant, external Zn²⁺ can prevent Ca²⁺ entry by inhibiting LTCC at concentrations as low as tens of nanomolars, which in turn suppresses the Ca²⁺-induced Ca²⁺ release from the SR [47102]. Zn²⁺ depresses the contractile force logarithmically with time, which seems to be related with dysregulation in calcium handling in electrically stimulated rat atrial cells [103] or smooth muscle cells [104]. In pre-diabetic rats, the improvement of diastolic relaxation by Zn²⁺ is attributed to a depressed LTCC inward current, phosphorylation of the ryanodine receptor and phospholamban, resulting in a reduced cellular Ca²⁺ load [89]. In guinea pigs, the administration of low concentrations of Zn²⁺ caused mainly sinus bradycardia, while higher concentrations of Zn²⁺ (30 µM) resulted in various types of arrhythmia that lead to cardiac asystole [105]. In humans, the cardiac ejection fraction increases with Zn²⁺ content [106], and Zn²⁺ supplementation to a cardioplegic solution reverses the loss of systolic function [27]. However, the importance of Zn²⁺ in cardiac muscle function is still incompletely understood.

In patients with ischemic cardiomyopathy, a higher copper level and a lower zinc level were observed [107]. Diabetic patients with congestive heart failure also exhibited a high concentration of urinary Zn²⁺ excretion [107]. Normally, the majority of plasma Zn²⁺ is associated with human serum albumin (HSA), but this interaction is allosterically disrupted by free fatty acids (FFAs), which eventually results in hypozincemia due to altered Zn²⁺ uptake in the cell [21]. Zinc deficiency is associated with enhanced PTP activity, which increases insulin receptor signaling and insulin resistance, which is a risk factor for cardiovascular disease development [108]. Theoretically, all conditions that lead to lower energy consumption by the heart could lead to at least temporary elevations of FFAs. Therefore, the possibility of a direct correlation between lower plasma Zn²⁺ levels and higher levels of FFAs cannot be overlooked, as it is evident in acute myocardial infarction [109] and chronic heart failure [110]. Zinc deficiency may also have deleterious effects in patients with heart failure, due to decreased intake secondary to decreased absorption, increased urinary loss through diuretics, or high levels of lipid peroxides as a result of oxidative stress [108]. However, paradoxically, heart failure medications, such as thiazide diuretics, angiotensin-converting enzyme inhibitors, angiotensin receptor blockers, and the diuretic furosemide can upset zinc metabolism, and subsequently lead to zinc deficiency [108111].

PKC is a central molecule in many signaling pathways and is particularly prominent in vascular smooth muscle and endothelial cells. As previously mentioned [58], alteration of PKC activity directed by the movement of Zn²⁺ modulates the action of multiple vasoactive factors that promote atherosclerotic events. Zn²⁺ may play a protective role in the maintenance of the integrity of endothelial cells [112] or the prevention of ultraviolet-induced skin damage [113]. Chronic zinc deprivation accelerates vascular smooth muscle cell proliferation [114], and in combination with calcification, may aggravate atherosclerosis progression [29].

Pancreatic β cells contain the highest amount of zinc among all cells within the human body. Zn²⁺ is vital for the precise handling of insulin in β-cells, from storage to the exertion of its effects. It has been suggested that the opening of ATP-sensitive K⁺ channels (K_ATP) in the pancreas is regulated by the activation of Zn²⁺ on both sides of the membrane, with potentially discrete sites of action located on the sulfonylurea receptor (SUR) subunit [115]. It has been recognized that zinc deficiency, or the aberrant expression of ZnT (e.g., ZnT8) or ZIP (e.g., ZIP4) proteins in the pancreas, is linked to the occurrence of pancreatic cancer and chronic pancreatitis [80]. Secreted Zn²⁺ exhibits autocrine or paracrine signaling in β-cells or neighboring cells. During glucose deprivation, Zn²⁺ serves as an intercellular signaling ion, and provides an "off-switch" for glucagon release from the pancreatic α-cells. Zn²⁺ also modulates the exocrine production of digestive enzymes and their role in nutrient absorption. Zinc's entry into the digestive tract is facilitated by enzyme-containing zymogen granules secreted from the acinar cells of the pancreas. The activity of zymogen is Zn²⁺-dependent, and ZnT2 aids in zinc transport, which eventually leads to digestive enzyme secretion [80].

Unlike other cells, prostate cells contain almost 35% of their total zinc in the cytoplasm, rather than sequester Zn²⁺ into vesicles and organelles. High zinc levels are emitted into the seminal liquid, assuming a critical role in sperm discharge and motility [116]. Antimicrobial properties have also been associated with Zn²⁺ in prostatic fluid and the gland itself. In cancer biology, evidence points to zinc's involvement in tumor suppression in several cancers [117]. Reduced levels of zinc in the prostate were also linked to prostate diseases, including benign prostatic hyperplasia and prostate carcinoma [80].

While zinc's role in mammary gland development [80] and remodeling is indisputable, the effects of its deficiency remain poorly understood. A single amino acid substitution in human ZnT2 reduces milk zinc concentration by about 75% and results in severe dermatitis and alopecia in the nursing infant [118]. Tissue expansion in the mammary gland is also mediated by Zn²⁺-dependent matrix metalloproteinases (MMPs). Again, the availability of cellular Zn²⁺ dictates the enzyme activity; therefore, a decrease in activity could result in impaired remodeling and subsequent breast disease.

Zinc deficiency is manifested by an immunocompromised status due to thymic atrophy, lymphopenia, or compromised cell- and antibody-mediated responses, which causes increased infection rates with prolonged durations [32119]. Zinc deficiency stimulates the production of cytokines, such as interleukin (IL)-1β, IL-2, IL-6, and tumor necrosis factor (TNF)-α, and the generation of these cytokines shows inverse responses to zinc supplementation [91]. Therefore, mild zinc deficiency with low-grade inflammation has been found in patients with atherosclerosis and diabetes mellitus [91]. In the allergic response, Zn²⁺ and its transporters are indispensable for Fc epsilon receptor I (FceRI)-mediated mast cell activation, since blocking or supplementation of zinc largely affects this allergic reaction through the involvement of degranulation and cytokine production [12]. In monocytes, Zn²⁺ affects differentiation by increasing cAMP and its guanosine analog (cGMP) through the inhibition of ACs and cyclic nucleotide phosphodiesterases [46120].