Nicotine and the taste allure for salty food

Smoking has been recognized as one agent that may decrease the effectiveness of the gustatory system to detect salt (Na) in foodstuffs. As a consequence, smokers tend to ingest saltier foods than nonsmokers. An increase in sodium intake has been associated with hypertension: thus, smoking may concur to the development of hypertension by impairing salt perception. Understanding the mechanisms underlying the action of smoking on salty taste represents the premise to design proper intervention aiming at restoring normal sensitivity to sodium in smokers. I addressed this issue by studying the effect of nicotine, one of the main components of tobacco smoke, on the sodium detection mechanism in rat taste cells. Electrophysiological analysis of these cells revealed that long-term exposure to nicotine reduced the ion current mediated by the Epithelial Sodium Channel (ENaC), one of the sodium receptors occurring in taste cells. As to the molecular mechanism responsible for such a current decrease, data were consistent with a reduction in the number of functional ENaCs in the membrane of taste cells. Therefore, nicotine reduces the capability of taste cells to respond to sodium ions. This might explain, at least in part, why smokers tend to use salt more abundantly when flavoring their food: they are just boosting the sensory information to be relayed to the brain.


Salt taste perception and smoking
Taste reception is a fundamental sensory activity for proper selection of food and beverages [1] .Among the several chemicals recognized by the taste system, sodium ions (Na + ) are of particular relevance.Na + represents the main cation of extracellular fluid and is a key factor in essential physiological processes undergoing in our body, from the generation and conduction of nerve impulses to the regulation of cell tonicity.Taste helps us to detect this mineral in foodstuffs: Na + elicits a specific sensation called salty taste that guides the ingestion of this cation (sodium intake) [2,3] .
Man evolved in low-sodium habitat and is well adapted to handle low salt availability.In modern life, however, salt (NaCl) is easily accessible and this has led to consumption beyond the physiological needs.Na + affects the extracellular fluid volume and therefore blood pressure: indeed, increase dietary sodium intake has been characterized as an important contributor to hypertension [4][5][6] .Hypertension is a major risk factor for many cardiovascular diseases including stroke, coronary heart disease, cardiac failure, and endstage renal disease.Recommendations to prevent the development of hypertension include a reduced sodium intake and avoiding

RESEARCH HIGHLIGHT
those factors, such as smoking, that may interfere with salt perception by taste system.
Smoking affects salt taste perception by raising the threshold for recognition and by reducing the perceived intensity at supra-threshold concentrations [7][8][9][10][11][12] .This means that smokers would consume more salt because they cannot taste it as well as nonsmokers, all things being equal.Indeed, smokers tend to ingest saltier foods than nonsmokers [13][14][15][16][17] , and this expose them to the risk of developing hypertension.The actual amount of sodium ions ingested by smokers can be higher than 4 g (200 mmol) a day [17] , that is, twice as much as the amount of sodium in a medium sodium diet (~100 mmol) [18] .An increase in sodium intake from ~1.5 g to ~3 g a day has been shown to induce an increase in the systolic blood pressure of ~8 mmHg [18,19] .Thus, smoking may concur to aggravate hypertension by acting on salt perception, that is, by reducing the effectiveness of the taste system to detect salt.

Nicotine action on sodium receptors in rat taste cells
The mechanisms underlying the action of smoking on salt taste perception are currently unknown.I have addressed this issue in my recent work [20] by testing the following hypothesis: nicotine acts at the level of taste cells by disrupting the sensory mechanism involved in sodium detection.I put forward this working hypothesis on the basis of the following considerations.
Nicotine, one of the main components of tobacco smoke, is a pharmacologically active alkaloid that affects several tissues [21] , including the peripheral sensory organs of taste, the taste buds [22][23][24][25][26] .Taste buds are made of taste cells, which detect chemicals dissolved in the saliva (including Na + ), transform them into electric impulses, and then relay sensory information to the brain [3,27] .I therefore reasoned that impairment (worsening) of salt taste perception induced by smoking could be explained, at least in part, in terms of nicotine affecting the mechanism involved in the detection of Na + in taste cells.
Increases in the sodium concentration in the oral cavity are thought to be transduced through a direct entry of Na + into taste cells via open channels located in their apical membrane bathed by saliva [27,28] .Among these, the Epithelial Na Channel (ENaC) represents one of the best characterized "sodium receptor" occurring in mammalian taste cells [29][30][31] .Thus, I hypothesized that nicotine could disrupt the functioning of this channel.
Active ENaCs can be monitored in single cell by analyzing the so-called response to amiloride with the electrophysiological technique of patch-clamp recording [32] .Amiloride is a diuretic drug that blocks ENaC in the sub-micromolar concentration range [27] .If a cell express functional ENaCs, application of amiloride to that cell causes a reduction in the ion current flowing through the membrane by shutting down these channels: this current reduction is referred to as the response to amiloride.I therefore tested whether nicotine affected the response to amiloride in taste cells of the rat, an animal model widely used in taste research.
To reproduce long term exposure to nicotine as found in smokers, drug was administered to rats via drinking water [25]   , allowing a continuous exposure [33] that resembles the one observed in habitual smokers [34,35] .The oral administration route is also similar to the "chewing tobacco" or the "nicotine gum" route of exposure in humans [10,36,37] .
The results of my research indicated that nicotine treatment exerted a clear-cut effect on taste cells by causing a significant decrease in the response to amiloride.This reduction could be explained in terms of different mechanisms.A reduced sensitivity to amiloride (expressed by the inhibition constant, Ki) would result in a reduced amplitude of the response to a given amiloride concentration.Similarly, a change in the ion selectivity (PNa/PK) of ENaCs in favor of potassium ions would cause a reduction in the response amplitude at any membrane potential.However, I found that both Ki and PNa/PK did not change after nicotine treatment.
It is well established that sensitivity of ENaC to amiloride inhibition depends on the subunit composition of the channel (which consists of three different subunits: , , and  [38] ).For example, variations in the expression level of the  or  subunit would have a profound impact on Ki [39] .Thus, my electrophysiological results indicated that chronic exposure to nicotine likely did not affect the subunit composition of ENaCs in taste cells.
Covalent modifications of the channel protein (such as phosphorylation by protein kinase A or ADP ribosylation) may induce changes in ENaC ion selectivity [40,41] .My results suggested that long-term exposure to nicotine unlikely induced covalent modification affecting PNa/PK ratio.
Assuming that the single-channel conductance of ENaC did not vary under nicotine treatment (as indicated by unchanged values for Ki and PNa/PK) [39,40] , then data could be interpreted on the basis of a reduction in the "activity" of the channels.Channel activity is defined as the number of functional channels (NENaC) times the channel open probability (PO) [42] .Of course, a decrease in channel activity could be due to a reduction in NENaC and/or to a decrease in PO.Nonetheless, a reduced activity of functional ENaCs would produce a smaller electrical signal when taste cells are stimulated by increasing the external sodium concentration.Therefore, a reduced activity of ENaCs would be reflected in taste cells being less sensitive to sodium variations in the saliva.
How nicotine affects the activity of ENaCs remains to be elucidated.However, some clues for the possible mechanism have been provided by other electrophysiological observations.I found that that membrane capacitance (Cm) was about 25% larger in nicotine-treated cells than in control ones.This effect was specific, because Cm did not change in taste cells lacking ENaCs.Cm is directly related to the actual extension of the membrane lipid bilayer, and therefore Cm changes could reflect heavy membrane remodeling.Variation in the rate of endocytosis and exocytosis may have a dramatic effect on cell membrane extension.ENaC is subjected to a continuous turnover in other cell types.Typically, the total cellular pool of ENaC turns over rapidly, with a half-life of about 40-120 min in cultured cells [43] .The density of functional ENaCs on the cell membrane is therefore the result of the equilibrium between channel exocytosis and endocytosis [43] .Nicotine induces endocytosis of ENaCs in human nasal epithelium cells in culture with concomitant reduction in membrane capacitance [44] .Thus my findings suggest that nicotine could interfere with regular ENaC membrane trafficking in taste cells, although at the moment it is difficult to reconcile an increase in ENaC endocytosis (which would explain the reduction in the response to amiloride) with an increase in membrane surface area of taste cells (increased Cm).

Restoring sodium taste sensitivity in smokers?
Findings on rat taste cells indicate that chronic administration of nicotine inhibits the activity of ENaC, which represents the "sodium receptor" [30] .Therefore, nicotine impairs the first molecular step in sodium detection.The biological consequence of this effect would be a reduced inflow of sodium ions into taste cells when sodium concentration increases in the saliva (as it occurs during chewing of salty food).Thus, taste cells would produce smaller biological signals in response to salt stimulation and this would be translated in reduced sensory input to nerve fibers.In short, the reduced capability of taste cells to detect sodium ions would impair the sensory analysis performed by taste system on salty food.
It is conceivable that a similar mechanism could explain, at least in part, the reduced taste sensitivity to sodium ions (salty taste) in smokers.Both amiloride-sensitive (ENaC-mediated) and amiloride-insenstive components have been described for sodium taste in humans [45][46][47][48][49][50][51][52][53] , although a great variability exists among individuals [52] .ENaC ,  and  subunits occur in human fungiform papillae [54,55] .On the basis of these evidences, it is tempting to speculate that the effect of nicotine on ENaC observed in rat taste cells could also take place in those individuals expressing the amiloride-sensitive sodium pathway.As a consequence, smokers would tend to add more salt in foods to compensate for a sodium detection mechanism partially disrupted by nicotine.
In conditions of reduced number of functional ENaCs, a simple intervention to restore (increase) sodium sensitivity of taste cells exposed to nicotine would be to enhance the activity of residual ENaCs.Several substances are known to act as positive modulators for these channels.For example, the amino acids arginine and lysine enhance currents through human ENaC expressed in Xenopus oocytes and enhance salt taste perception in human subjects [55] .Therefore, adding a positive modulator of ENaCs to table salt could be a possible remedy to correct the sensory deficit in salt perception caused by nicotine.

Conflicting interests
The author has declared that no conflict of interests exist.