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. Author manuscript; available in PMC: 2016 Dec 12.
Published in final edited form as: Can J Physiol Pharmacol. 2009 Oct;87(10):821–830. doi: 10.1139/Y09-072

Protein kinase inhibition differentially regulates organic cation transport1

Alexander M Gerlyand 1, Daniel S Sitar 2,2
PMCID: PMC5153330  CAMSID: CAMS1166  PMID: 20052008

Abstract

Previous studies showed that amantadine transport increased while tetraethylammonium (TEA) transport decreased in kidney tissue from diabetic rats. Changes in transport activity were reversed by exogenous insulin. We hypothesized that this difference in transport regulation is due to differential regulation of different transport systems. Native human embryonic kidney cortex cells (HEK293 cell line) and rat organic cation transporter (rOCT)-transfected cells were used to test the hypothesis. In support of differential regulation, short-term glucose starvation stimulated amantadine transport and inhibited TEA transport, but the effect was bicarbonate-modulated only for amantadine. cAMP analogues inhibited TEA transport while stimulating amantadine transport. This effect was additive to the effect of insulin, and the presence of bicarbonate affected the extent of the change. Our findings indicated that regulation of rOCT 1 and 2 was mediated by transmembrane adenylyl cyclase, and regulation of amantadine transport was mediated by soluble adenylyl cyclase, suggesting that intracellular microdomains of cAMP may be important in determining overall cellular transport for organic cations. Soluble adenylyl cyclase activity is known to be modulated by bicarbonate and lactate. These observations support our hypothesis and reconcile our previous studies demonstrating increased transport affinity for amantadine in the presence of bicarbonate and decreased transport affinity in the presence of lactate.

Keywords: organic cation transport, amantadine, tetraethylammonium, rOCT1, rOCT2, HEK293 cells, kidney

Introduction

Organic cation transporters (OCTs) are members of a family of membrane-associated proteins (SLC22) that serve to move positively charged xenobiotics (drugs, environmental toxins) and endogenous metabolites within the body from the peritubular blood to the tubular lumen for elimination by the kidney into urine (Jonker and Schinkel 2004). Two organic cationic probes, amantadine and tetraethylammonium (TEA), characterize distinct portions of the organic cation transport system (Goralski and Sitar 1999; Goralski et al. 2002; Wright et al. 2004). Gründemann et al. (1994) were the first to identify a polyspecific OCT, rOCT1, in the rat kidney through expression cloning. To date numerous OCTs have been cloned. In the rat, rOCTs 1–3 possess substrate specificity for TEA (reviewed by Burckhardt and Wolff 2000). Of those cloned, rOCT1 and rOCT2 are prominent forms in the adult rat kidney and mediate organic cation transport at the basolateral membrane of proximal cells (Koepsell et al. 1999; Sweet et al. 2000; Urakami et al. 1998). The identity of the amantadine transporter(s) remains unresolved (Goralski et al. 2002; Wright et al. 2004).

Both protein kinase A (PKA) and PKC have been implicated in the regulation of OCTs (Ciarimboli and Schlatter 2005; Ciarimboli et al. 2005). Cyclic adenosine monophosphate (cAMP) is a secondary messenger involved in the early response to myriad extracellular and metabolic signals in virtually every living cell. In kidney cells, this signalling molecule has been implicated in the regulation of OCTs through its activity on PKA (Cetinkaya et al. 2003; Mehrens et al. 2000; Pietig et al. 2001). Although there is growing information on the functional characteristics and localization of OCTs, many questions remain unsolved (Berkhin and Humphreys 2001; Popp et al. 2005; Schmitt and Koepsell 2005; Volk et al. 2003). Evidence for their regulation is seen through perturbation of drug transport systems in diabetes, the so-called renal protection phenomenon in patients and animal models, and reflected by the apparently reduced nephrotoxicity to gentamicin, cephaloridine, and cisplatin that is attributed to decreased drug accumulation in the renal cortex (Ramsammy et al. 1987; Scott et al. 1990; Teixeira et al. 1982; Valentovic et al. 1989). Further instances of this perturbation have been demonstrated by amantadine and TEA, with one notable difference: amantadine transport increased while TEA transport decreased in kidney tissue from diabetic rats compared with that from control rats (Goralski et al. 2001; Grover et al. 2002). For both substrates, transport perturbations were reversed by exogenous insulin, implicating the diabetic state as the responsible mechanism. Thus, there may be differential regulation between the transport systems. A recent study provided molecular support for the observations in diabetic rats by demonstrating reduced expression of rOCT1 and rOCT2 in the diabetic state, and a restoration in their levels by exogenous insulin (Grover et al. 2004). The complete mechanism for this observed differential perturbation of organic cation transport is unclear. For the purposes of our studies, the diabetic state has served as a tool to investigate further factors behind that regulation.

Identification of a cytosolic soluble adenylyl cyclase (sAC) (Chen et al. 2000) in addition to the classical transmembrane adenylyl cyclase (tmAC) enabled the hypothesis that complex signalling networks may be created by unique cAMP signalling domains (Zippin et al. 2001). A key characteristic of sAC is its unique regulation by bicarbonate, a major catabolic end-product of energy metabolism. Our previous reports, demonstrating bicarbonate dependence of amantadine transport (Escobar and Sitar 1995; Escorbar et al. 1994), and other studies suggesting the modulation of TEA transport by bicarbonate (Goralski et al. 2002; Ullrich et al. 1991) could be reconciled by the presence of unique cAMP signalling domains.

In this report, we provide evidence that the mechanisms behind perturbation of organic cation transport systems in diabetes are tied to cAMP pools generated from tmAC and sAC. Central to these studies is the discovery by Goralski et al. (2002) that native human embryonic kidney cortex cells (HEK293 cell line) transport amantadine and not TEA. Transfecting native HEK293 cells with rOCT1 or rOCT2 enabled us to study OCT transporter interaction with TEA as a probe. Our studies indicate additive effects exist between insulin and cAMP analogs in modulating transport. More domain-specific forskolin/bicarbonate/protein kinase inhibition studies suggest that sAC pools modulating amantadine energy-dependent transport are strongly influenced by energy levels in the cell, in contrast to rOCT1 and rOCT2 modulation, which appear to be primarily influenced by tmAC.

Materials and methods

Cell culture

The HEK293 cell lines stably transfected with rOCT1 or rOCT2 were a generous gift from the laboratory of Dr. Hermann Koepsell (Institute of Anatomy and Cell Biology, Würzburg, Germany). Nontransfected HEK293 cells (American Type Culture Collection, Manassas, USA) and HEK293 cells stably transfected with rOCT1 and rOCT2 (pRc-CMV; Invitrogen, Groningen, The Netherlands) were grown to 90% confluency at 37 °C in a 95% O2/5% CO2 humidified atmosphere in 175 mL culture flasks (Corning, New York, USA) in low-calcium, low-glucose Dulbecco’s modified Eagle’s medium (DMEM), supplemented with 44 mmol/L NaHCO3, 10% (v/v) foetal bovine serum (Gibco/Invitrogen, Grand Island, USA), and 0.3 mg/mL geneticin (Gibco/Invitrogen). Monolayers were trypsinized with Ca2+- and Mg2+-free phosphate-buffered saline (PBS) and 0.25% trypsin-EDTA (Gibco/Invitrogen). PBS contained 2.7 mmol/L KCl, 1.5 mmol/L KH2PO4, 136.9 mmol/L NaCl, and 1.6 mmol/L NaHPO4·7H2O (pH 7.4). The flasks were then used to seed 75 mL culture flasks (Corning) with 5 million cells in 10 mL medium (for long-term and short-term treatment experiments), and 175 mL flasks with 12.5 million cells in 25 mL medium (for time course and glucose starvation experiments). The medium was changed daily. Cells for uptake studies were used on day 2–3 of growth when monolayers achieved 90% confluency.

Amantadine and TEA uptake assays

Uptake studies were performed with suspended cells in bicarbonate-free Cross-Taggart (CT) buffer, or in bicarbonate-containing Krebs–Henseleit solution (KHS) buffer. CT contained (in mmol/L) 135 NaCl, 4.7 KCl, 1.2 MgCl2, 1.4 KH2PO4, 15 sodium phosphate buffer, 1.0 CaCl2, and 11 glucose (pH 7.4). KHS contained (in mmol/L) 125 NaCl, 4.7 KCl, 1.2 MgCl2, 1.4 KH2PO4, 25 NaHCO3, 2.5 CaCl2, and 11 glucose (pH 7.4). For glucose starvation experiments, glucose (11 mmol/L) was exchanged for mannitol in the respective buffers, and equal osmolality was confirmed using a microosmometer (μOsmette, Precision Systems, Natick, USA). For all transport assays in KHS, the pH and pCO2 levels of the buffer were adjusted by bubbling with O2 and CO2 (95%:5%). For bicarbonate-free conditions, sealed flasks were used to maintain CT buffer under bicarbonate-free conditions. Viability of the cells was confirmed using trypan blue staining. Before each assay, medium was removed from the cells; attached cells were washed with PBS and replaced with fresh buffer containing insulin (0.8–20 mU/mL), 8-bromoadenosine 3′,5′-cyclic monophosphate sodium salt (8-Br-cAMP) (100 μmol/L), N6,2′-O-dibutyrylade-nosine 3′,5′-cyclic monophosphate sodium salt (dibutyryl cAMP) (100 μmol/L), forskolin (1–10 μmol/L), and staurosporine (6 nmol/L) and allowed to incubate at 37 °C in an incubator (Thermo Forma Series II water jacketed) for 30 min for insulin and cAMP analogues and 60 min for forskolin and staurosporine. Forskolin and staurosporine were dissolved in dimethyl sulfoxide (DMSO). Final DMSO concentration in CT or KHS buffer was 0.5% v/v. The control incubation contained 0.5% v/v DMSO in CT or KHS buffer solution. For long-term treatment of cells, appropriate reagents were prepared in fresh DMEM medium that was used to replace the original medium on subconfluent cultures 24 h before the experiments.

Each flask was then rinsed with bicarbonate-free CT buffer or bicarbonate-containing KHS buffer (15 mL). Cells were detached by scraping in the presence of 15 mL of buffer followed by centrifugation (IEC model Centra-4 centrifuge) at 1000g for 10 min. The pelleted cells were resuspended in 810 μL of buffer and placed in a water bath (25 °C) with shaking (100 oscillations/min) until ready for use. For glucose starvation and time course studies, the cells were resuspended in 3 mL of buffer. Cells, 90 μL, final protein content 4–6 mg/mL as measured by the biuret assay (Gornall et al. 1949), were placed in microcentrifuge tubes in a water bath at 25 °C with shaking (100 oscillations/min). [14C]TEA (10 μL, 20 μmol/L final experiment concentration) or [3H]amantadine (10 μmol/L final experiment concentration) was added to the wall of the centrifuge tube. The transport reaction was started by vortex mixing and placed in a water bath (25 °C) with shaking (100 oscillations/min) for the appropriate time period. The reaction was stopped with 1 mL of ice-cold stopping buffer (10 μmol/L quinine in KHS or CT). The tubes were then centrifuged for 1 min at 13 000g (Fisher, model 235A) and the supernatant was discarded. The cells were washed and centrifuged twice with 1 mL of the stopping buffer. Pellets were then dissolved in 200 μL of Triton X-100 (J.T. Baker, Philipsburg, USA) (0.1% v/v), placed into scintillation vials containing 4 mL of Ready Safe scintillation fluid (Beckman Instruments, Fullerton, USA), and counted in a Beckman model LS5801 scintillation counter.

Chemicals

[3H]Amantadine (28 Ci/mmol) (1 Ci = 3.7 × 1010 Bq) was obtained from Amersham International (Buckinghamshire, UK). [14C]TEA (55 mCi/mmol) was obtained from American Radiolabeled Chemicals (St. Louis, USA). Unlabeled amantadine was obtained from DuPont Canada (Mississauga, Canada). All standard chemicals, forskolin, 8-Br-cAMP, dibutyryl-cAMP, staurosporine, quinine, and TEA were obtained in highest available purity from Sigma Chemical (St. Louis, USA). Human biosynthetic regular insulin (100 U/mL) was a gift from Novo Nordisk. All other chemicals were of the highest grade available from commercial suppliers.

Data analysis

The 3H and 14C measurements from the liquid scintillation counter and the protein determinations for the amantadine and TEA uptake assays were used to calculate total uptake into the cells. Total uptake is reported as specific uptake (nonspecific uptake subtracted) of amantadine or TEA by the HEK293 cells (in nmol/mg protein in 10 min). In each experiment, triplicates were used for each treatment and the mean result was used. Data are shown for 3–6 independent experiments, as indicated by the n values. Specific uptake was calculated as the difference between uptake of [3H]amantadine and [14C]TEA in the absence and presence of 10 μmol/L quinine (the standard OCT inhibitor).

Data for [3H]amantadine and [14C]TEA uptake were analysed using a one-factor analysis of variance (ANOVA) followed by the Tukey post hoc comparison among means (Prism 3, GraphPad software, San Diego, USA). For glucose starvation experiments, the paired two-sided Student’s t test was used to evaluate statistical significance of the effects. One-way ANOVA was used for all of the data to determine whether a buffer effect existed between the groups. The appropriate model ANOVA was used for all other comparisons. A two-tailed probability level of 0.05 or less was used for statistical significance. Results were expressed as means ± SE.

Results

Time course of TEA uptake in rOCT1- or rOCT2-transfected HEK293 cells, and amantadine uptake in native HEK293 cells

Amantadine transport displayed characteristic bicarbonate dependence of initial rate and intracellular steady-state concentrations, whereas TEA uptake by rOCT1 and rOCT2 was not bicarbonate dependent (Figs. 1A–1C). Initial uptake rates and intracellular steady-state concentrations were correlated, indicating that steady-state concentrations are determined by steady-state uptake mediated by the OCTs. Subsequently, a 10 min incubation time, reflecting steady-state uptake, was used to measure effects on regulation of organic transporter activity.

Fig. 1.

Fig. 1

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Time course of TEA uptake in (A) rOCT1-transfected HEK293 cells, (B) rOCT2-transfected HEK293 cells, and amantadine uptake in (C) native HEK293 cells. Data are means ± SE (n = 3).

Regulatory trends for amantadine versus rOCT1 or rOCT2 transporter systems after 24 h incubation with insulin and low/high glucose

Preincubation with insulin (10 mU/mL) decreased TEA transport by rOCT1 in all 3 treatment groups and was unaffected by high glucose or bicarbonate (Fig. 2A). In rOCT2-transfected cells, preincubation with insulin suggested a trend for decreased TEA transport that did not achieve statistical significance (Fig. 2B). In contrast, treatment of native HEK293 cells with insulin increased amantadine transport by 72% in a dose-dependent manner, regardless of glucose or buffer conditions (Fig. 3). Amantadine uptake was maximal in the presence of bicarbonate and inhibited at least 50% in its absence. Pretreatment of native HEK293 cells with a high concentration of glucose (25 mmol/L), alone or together with insulin, inhibited amantadine transport by 67% regardless of the presence of insulin. These data provide the first direct evidence of differential regulation of the organic cation transport system by insulin and glucose in a cell culture model.

Fig. 2.

Fig. 2

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The effect of insulin and high glucose preincubation (24 h) on high-capacity TEA uptake in (A) rOCT1-transfected HEK293 cells (n = 5) and (B) rOCT2-transfected HEK293 cells (n = 4). Data are means ± SE. *, significant at p < 0.05 and **, p < 0.01 compared with the respective control observation.

Fig. 3.

Fig. 3

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The effect of insulin and high glucose pre-incubation (24 h) on high-capacity amantadine uptake in native HEK293 cells (n = 4). Data are means ± SE. *, significant at p < 0.05 and **, p < 0.01 compared with the respective control observation; ††, p < 0.01 between treatment groups compared with 5.6 mmol/L glucose KHS buffer treatment.

Acute glucose starvation and modulation of organic cation transport in HEK293 cells

Cells were exposed to a 30 min acute glucose starvation in CT and KHS buffers, where glucose was replaced with iso-osmotically equivalent mannitol. In both rOCT1- and rOCT2-containing cells, a 50% reduction in TEA uptake compared with controls was observed, regardless of the presence of bicarbonate (Figs. 4A, 4B). In contrast, amantadine uptake increased in glucose-starved cells compared with control cells in both the absence and presence of bicarbonate (45% and 49%, respectively) (Fig. 4C). By using the Trypan blue exclusion assay, cell viability of approximately 98% up to 3 h was demonstrated in both the absence and presence of glucose in CT and KHS buffer (data not shown). The effect of glucose starvation on amantadine transport in the presence of bicarbonate is greater than in its absence.

Fig. 4.

Fig. 4

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The effect of acute glucose starvation (30 min) on high-capacity TEA uptake in A) rOCT1-transfected HEK293 cells (n = 4), B) rOCT2-transfected HEK293 cells (n = 4), and amantadine uptake in C) native HEK293 cells (n = 4, CT; n = 5, KHS). Data are means ± SE. *, significant at p < 0.05, **, p < 0.01, and ***, p < 0.001 compared with the respective control observation; ††, p < 0.01 and †††, p < 0.001 between buffer treatment groups.

cAMP as a metabolic signal for insulin modulation of organic cation transport

Incubation of rOCT1-transfected cells with Br-c-AMP or dibutyryl c-AMP (100 μmol/L) in the presence and absence of insulin (20 mU/mL) for 30 min before the transport studies resulted in the inhibition of TEA uptake compared with control in both CT and KHS buffers (Fig. 5). An additive effect of insulin to Br-cAMP and dibutyryl-cAMP in rOCT1-transfected cells occurred only in the presence of bicarbonate. In rOCT2-transfected cells, treatment with c-AMP analogues alone caused a marked decrease in TEA uptake in KHS; with insulin this effect was additive, but once again only in the presence of bicarbonate (Fig. 6). One-way ANOVA of data from rOCT2-containing cells confirmed the presence of a buffer effect between the 2 insulin-treated groups. Evidence of an effect of insulin and dibutyryl-cAMP compared with analogue alone was demonstrated in KHS. Examination of the effect of insulin and increased c-AMP analogue levels on amantadine transport demonstrated an increase in amantadine uptake only when insulin and the cAMP analogue were combined (Fig. 7). One-way ANOVA of amantadine transport data in native HEK293 cells confirmed the presence of a buffer effect among all respective treatments. Treatment of the cells with insulin in addition to c-AMP analogue resulted in stimulation of uptake compared with c-AMP analogue alone. These results indicate that bicarbonate and cAMP analogues alone and together with insulin may acutely modify OCT regulation. Evidence of additive effects among those components in the regulation of rOCT1, rOCT2 and amantadine transport suggests a complementary mechanism of action that appears to be metabolic in origin.

Fig. 5.

Fig. 5

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The effect of acute treatment (30 min) of insulin (20 mU/mL) and cAMP analogues (100 μmol/L) on high-capacity TEA uptake in rOCT1-transfected HEK293 cells (n = 4). Data are means ± SE. *, significant at p < 0.05, **, p < 0.01, and ***, p < 0.001 compared with the respective control observation; ‡‡, p < 0.01 between respective cAMP analogue treatments.

Fig. 6.

Fig. 6

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The effect of acute treatment (30 min) of insulin (20 mU/mL) and cAMP analogues (100 μmol/L) on high-capacity TEA uptake in rOCT2-transfected HEK293 cells (n = 5, CT; n = 4, KHS). Data are means ± SE. **, significant at p < 0.01 and ***, p < 0.001 compared with the respective control observation; †, p < 0.05 between buffer treatment; ‡, p < 0.05 between respective cAMP analogue treatments.

Fig. 7.

Fig. 7

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The effect of acute treatment (30 min) of insulin (20 mU/mL) and cAMP analogues (100 μmol/L) on high-capacity amantadine uptake in native HEK293 cells (n = 4, CT; n = 5, KHS). Data are means ± SE. *, significant at p < 0.05 and **, p < 0.01 compared with the respective control observation; †, p < 0.05, ††, p < 0.01, and †††, p < 0.001 between buffer treatments; ‡‡, p < 0.01 between respective cAMP analogue treatments.

Evaluation of the contributions of intracellular cAMP pools from tmAC and sAC using forskolin and bicarbonate to perturb organic cation transport

Pre-incubation with 1 or 10 μmol/L forskolin for 60 min was followed by uptake experiments examining [14C]-TEA and [3H]-amantadine uptake. In rOCT1-containing cells, a 30% reduction in TEA uptake was observed after preincubation with 10 μmol/L forskolin in CT buffer (Fig. 8). In KHS buffer, rOCT1-containing cells demonstrated a 22% reduction in TEA uptake after preincubation with 1 μmol/L and 10 μmol/L forskolin. Protein kinase inhibition by staurosporine alone and in addition to PKA activation by 10 μmol/L forskolin resulted in inhibition of TEA uptake compared with control, with no differences between the 2 treatments in both buffers. In rOCT2-containing cells, a 20% reduction in TEA uptake was observed after forskolin pretreatment in CT buffer (Fig. 9). Protein kinase inhibition by staurosporine alone and in addition to cAMP-dependent PKA activation by 10 μmol/L forskolin resulted in inhibition in TEA uptake in both buffers, with no differences between the 2 treatments. One-way ANOVA confirmed the existence of a buffer effect between the 10 μmol/L forskolin treatment groups. Contrary to the above findings, in HEK293 cells we observed no effect of forskolin on amantadine uptake in the presence or absence of bicarbonate (Fig. 10). Staurosporine alone and with forskolin resulted in the stimulation of amantadine uptake, but only in CT buffer. Further analysis of the data confirmed the existence of a buffer effect between respective treatment groups. These data suggest that the cAMP pools generated by tmAC stimulation with forskolin may affect only rOCT1 and rOCT2 activity. On the other hand, the presence of bicarbonate in the buffer, through its action on sAC, may contribute to the cAMP pools primarily modulating that portion of the organic cation transport system identified by the cationic marker amantadine, and not TEA.

Fig. 8.

Fig. 8

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The effect of acute (60 min) forskolin treatment on high-capacity TEA uptake in rOCT1-transfected HEK293 cells (n = 4). Data are means ± SE. *, significant at p < 0.05 and ***, p < 0.001 compared with the respective control observation.

Fig. 9.

Fig. 9

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The effect of acute (60 min) forskolin treatment on high-capacity TEA uptake in rOCT2-transfected HEK293 cells (n = 4). Data are means ± SE. *, significant at p < 0.05 and ***, p < 0.001 compared with the respective control observation; †, p < 0.05 between buffer treatments.

Fig. 10.

Fig. 10

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The effect of acute (60 min) forskolin treatment on high-capacity amantadine uptake in native HEK293 cells (n = 4). Data are means ± SE. **, significant at p < 0.01 compared with the respective control observation; ††, p < 0.01 and †††, p < 0.001 between buffer treatments.

Discussion

It is widely believed that understanding the regulation of the organic cation transport system will improve our ability to better understand the consequences of its perturbations in the renal elimination of drugs with cationic characteristics and in the accumulation of these kind of drug substrates by other tissues, for example, liver. The evidence of differential regulation of rOCT1 and rOCT2 transport of TEA compared with the transport of amantadine by insulin and cAMP in our studies suggests that the regulatory processes and consequences for drug excretion and tissue uptake are likely to be complex. The apparent differential roles for tmAC and sAC and the selective effect of bicarbonate on sAC activity infer a complex homeostatic regulatory process.

The effect of metabolism on organic cation transport via energy generation in the form of ATP and its concentration gradients has been well demonstrated (Steen et al. 1991; Inui et al. 2000). Diabetes characterization by alterations in levels of glucose and insulin, and their attribution to the development of diabetes-specific microvascular pathology (The Diabetes Control and Complications Trial Research Group 1993; UK Prospective Diabetes Study (UKPDS) Group 1998), serve to link metabolism to the normal functioning of the organic cation transport system. The release of cAMP as an important regulator of metabolism, controlling both glucose release and glycogen synthase activity through the action of PKA, and the discovery of the metabolic sensor sAC implicate cAMP as a likely candidate for modulating organic cation transport in diabetes.

The differential regulation evidenced consistently in our studies between the amantadine transporter system and the TEA-selective rOCT1 and rOCT2 transporters by insulin and cAMP is in agreement with results from 2 previous studies in renal proximal tubules and kidney cortex slices of diabetic rats showing amantadine transport and TEA accumulation also to be differentially regulated in the disease condition (Goralski et al. 2001; Grover et al. 2002). The mechanism to explain our observation of the absence of an acute insulin effect (30 min) for all 3 transporters (Figs. 4A, 4B) and the presence of one for amantadine and rOCT1 during 24 h of incubation (Fig. 2) remains to be explored further; however, it may be that insulin’s effects on OCTs are also transcriptional, translational, and (or) posttranslational (i.e., glycosylation) in origin and therefore require additional time to evolve to functionally significant degrees.

Apparent additive effects among the roles of insulin and cAMP in regulating organic cation transport may be related to cAMP microdomains. Typically, insulin has been shown to decrease whole-cell cAMP levels in several tissues (Butcher et al. 1966; Butcher and Sutherland 1967; Blecher et al. 1968) with the exception of the thyroid, where it has been shown to increase them (Dremier et al. 2002). Our experimental observations of additive effects between insulin and cAMP may be explained by several factors. First, it should be recalled that interpretation of changes in whole-cell levels of cAMP are not indicative of levels in localized cellular domains (Rich et al. 2000). Second, there is evidence that both insulin and insulin-like growth factor 1 (IGF-1) receptors form functional hybrids (Patti and Kahn 1998; Butler and LeRoith 2001), and both possess the capacity to achieve the same biological endpoints by different signalling systems (Miele et al. 2000). In contrast to insulin’s typically reported relationship to the cAMP signal, IGF-1’s relationship is reported to be that of potentiation both at the genetic and cellular protein level (Grellier et al. 1996). Whether we are looking at insulin receptor, IGF-1 receptor, or the activity of both still remains to be determined; nonetheless, cAMP signalling networks allow room for interpretational complexity. Reports indicating the involvement of cAMP in all 3 glucose-sensing systems in yeast (Thevelein and de Winde 1999; Dumortier et al. 2000; Rolland et al. 2000) serve to corroborate our ability to manipulate cAMP levels via glucose in the HEK293 cell culture model. A high glucose concentration environment indicated the absence of a hyperglycemic effect on rOCT1 and rOCT2 transporter function (Fig. 2) and allowed separation from its relationship to glycemic effects on the cell, at least in our experimental model. Energy-dependent amantadine transport on the other hand is modulated by both insulin and glucose (Fig. 3). However, effects are most likely independent from one another, since the proportional increase in transporter activity by respective insulin concentrations is consistent among treatment groups, regardless of the presence of elevated glucose or bicarbonate in the medium. It may be that rOCT1 and rOCT2 are not as intrinsically tied to metabolism or cytoplasmic events as we observe in respect to the amantadine transporter. Hyperglycemia is generally characterized by increases in intracellular ATP levels through stimulation of oxidative phosphorylation (Brownlee 2001). Its effect on amantadine transport may be explained by the increased anaerobic glycolysis associated with an overloading of aerobic metabolism. The consequence is increased lactate accumulation, which has been documented to inhibit amantadine transport (Escobar et al. 1995). Acute glucose starvation in a glucose-free buffer system in contrast to hyperglycemia has been characterized by the ability to lower intracellular ATP concentrations (Nilius and Droogmans 2001). Acute removal of glucose from the extracellular environment of the cell generates a strong positive stimulatory signal for metabolism to generate the necessary fuel for cellular function through PKA-stimulated glucose transporters and glycogen synthase activity, both of which are mediated by cAMP. Congruent with expectations of elevated cAMP levels, examination of the effect of glucose removal on these transport systems indicated strong inhibition of the uptake of TEA in rOCT1 and rOCT2 transfected cells, while stimulating amantadine uptake in native HEK293 cells (Fig. 4). Our manipulations of the glucose-sensing machinery implicate cAMP microdomains behind this observed regulation.

The involvement of sAC yields new pathways of signalling that tie the end-product of catabolism, CO2, directly into the function of an array of physiological processes through cAMP. A report that insulin increases pHi in a dose-dependent manner, with no change in extracellular pH in the kidney tissue of rabbits (Takahashi et al. 1996), reinforces the ability of insulin to generate HCO3− within our cell culture system. The ability of CO2, bicarbonate, and insulin to modulate cAMP appears to intrinsically link them in terms of regulatory control. Increased amantadine transport in the presence of additive effects of cAMP analogue and insulin is consistent with reports demonstrating its strong connection to energy dependence and to its inhibition by the anaerobic end-product lactate. In the instance of a diffusion model, cAMP analogues would probably exert an effect on effectors located at the level of the plasma membrane first, the cytosol second, and the nuclear membrane last. We used forskolin and bicarbonate in an effort to determine forskolin-sensitive tmAC and bicarbonate-sensitive sAC contributions, respectively, to the cAMP pools modulating organic cation transport. This strategy, we believe, allowed us to generate more specific cAMP signals compared with the large-scale nonspecific cAMP elevations generated by the analogues alone. What we are able to differentiate is that rOCT1 and rOCT2 appear to be regulated primarily by tmAC sources of cAMP, at least in our model system. There is some suggestion for bicarbonate modulation of rOCT2 transport. A possible explanation may be the existence of 2 isoforms of PKA (I and II), whose phosphorylation of target proteins exhibits biphasic responses to cAMP concentrations (Dyer et al. 2003). The amantadine transport system, on the other hand, is unaffected by tmAC-generated cAMP pools and is solely modulated by the bicarbonate in the buffer. Our decision to use staurosporine as our protein kinase inhibitor was a result of indications in literature of crosstalk among the PKC and PKA systems. Since PKC stimulation has been reported in the literature to cause a depression in rOCT transporters, we used a concentration of staurosporine reported to inhibit both PKA and PKC. We observed differential regulation between the 2 transport systems. Inhibition of the protein kinase systems further depressed rOCT1 and rOCT2 function, but enhanced stimulation of the amantadine transporter.

In summary, our present study has provided evidence for putative mechanisms by which experimental diabetes can differentially modulate organic cation transport via effects on tmAC and sAC. The influence of insulin on energy levels within the cell, through its effects on glucose metabolism, subsequent CO2 generation, extracellular and intracellular HCO3− formation, and hence sAC-derived cAMP, may contribute to the baseline cAMP signals modulating organic cation transport.

Acknowledgments

This study was funded by grants from the Canadian Institutes of Health Research (ROP14710) and the Manitoba Health Research Council.

Abbreviations

sAC

soluble adenylyl cyclase

tmAC

transmembrane adenylyl cyclase

cAMP

cyclic adenosine monophosphate

ANOVA

analysis of variance

8-Br-cAMP

8-bromoadenosine 3′,5′-cyclic monophosphate

CT

Cross-Taggart

dibutyryl cAMP

N6,2′-O-dibutyryladenosine 3′,5′-cyclic mono-phosphate

DMEM

Dulbecco’s modified Eagle’s medium

DMSO

dimethyl sulfoxide

HEK

human embryonic kidney

IGF-1

insulin-like growth factor 1

KHS

Krebs–Henseleit solution

rOCT

rat organic cation transporter

PBS

phosphate-buffered saline

PKA

protein kinase A

TEA

tetraethylammonium

Footnotes

1

This article is one of a selection of papers published in a special issue celebrating the 125th anniversary of the Faculty of Medicine at the University of Manitoba.

Contributor Information

Alexander M. Gerlyand, Department of Pharmacology and Therapeutics, University of Manitoba, A220–753 McDermot Avenue, Winnipeg, MB R3E 0T6, Canada

Daniel S. Sitar, Department of Pharmacology and Therapeutics, University of Manitoba, A220–753 McDermot Avenue, Winnipeg, MB R3E 0T6, Canada; Department of Internal Medicine, and Department of Pediatrics and Child Health, Faculty of Medicine, Centre on Aging, and Faculty of Pharmacy, University of Manitoba, Winnipeg, MB R3E 0T6, Canada.

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