Iriomoteolides: novel chemical tools to study actin dynamics.

Despite its promising biological profile, the cellular targets of iriomoteolide-3a, a novel 15-membered macrolide isolated from Amphidinium sp., have remained unknown. A small library of non-natural iriomoteolide-3a analogues is presented here as a result of a novel, highly convergent, catalysis-based scaffold-diversification campaign, which revealed the suitable sites for chemical editing in the original core. We provide compelling experimental evidence for actin as one of iriomoteolides' primary cellular targets, establishing the ability of these secondary metabolites to inhibit cell migration, induce severe morphological changes in cells and cause a reversible cytoplasmic retraction and reduction of F-actin fibers in a time and dose dependent manner. These results are interpreted in light of the ability of iriomoteolides to stabilize F-actin filaments. Molecular dynamics simulations provide evidence for iriomoteolide-3a binding to the barbed end of G-actin. These results showcase iriomoteolides as novel and easily tunable chemical probes for the in vitro study of actin dynamics in the context of cell motility processes including cell invasion and division.

Interactions of side (left and right ovary) with the number of follicles per ovary and with the intraovarian relationships between dominant follicle and corpus luteum in heifers.

The interactions between side of ovary (left ovary [LO] and right ovary [RO]) and number of follicles per ovary and between side and intraovarian patterns were studied in heifers with two follicular waves (anovulatory wave 1 and ovulatory wave 2). Intraovarian patterns were on the basis of location of the dominant follicle (DF) and corpus luteum (CL) and were termed DF-CL, DF, CL, and devoid. The frequency of the DF-CL intraovarian pattern was greater for the RO than for the LO in wave 1 (80 of 121; P < 0.0004) and in wave 2 (54 of 83; P < 0.006). For each wave, the DF of the DF-CL and DF patterns was more often in the RO for the ipsilateral relationship (e.g., wave 1: 66% vs. 48%; P < 0.01) and in the LO for the contralateral relationship (52% vs. 34%; P < 0.01). An interaction between side and pattern (P < 0.05) for number of follicles in wave 2 that attained 6 mm was from a greater number in RO than in LO when a DF was present (DF-CL and DF patterns). An interaction of side and pattern for the number of wave 2 regressing subordinate follicles that recovered (increased in diameter) and became part of the subsequent wave 1 was greater (P < 0.05) for LO than for RO for the DF pattern but not for the CL pattern. An effect of side or an interaction that involved side was not found for the greater dimensions and blood flow for both the DF and CL of the DF-CL pattern. Results indicated that side interacted with ovarian pattern for number of DF-CL patterns, side of DF, number of follicles per ovary, and recovery of regressing wave 2 follicles. The hypothesis was supported that some aspects of follicle dynamics reflect an interaction of side and intraovarian pattern. Future studies on the effect of side on luteal or follicle dynamics could be incomplete or misleading if intraovarian patterns are ignored.

Stimulation of regressing subordinate follicles of wave 2 with a gonadotropin product in heifers.

The recovery of regressing wave-2 subordinate follicles was studied by treating heifers with a gonadotropin product that had about 84% and 16% of follicle-stimulating hormone and luteinizing hormone activity, respectively. A treated group (n = 8) received a single dose of 50 mg (2.5 mL) of the gonadotropin product, and a control group (n = 8) received 2.5 mL of saline vehicle. The group assignment of heifers was not known to the ultrasonographer who tracked the follicles and measured follicle diameters. Follicle measurements began on the day of expected follicle deviation in wave 2 (largest follicle closest to 8.5 mm), and treatment (hour 0) was given on Day 13.4 ± 0.2 (Day 0 = ovulation) when the dominant follicles of waves 1 and 2 were 14.1 ± 0.3 mm and 10.7 ± 0.1 mm, respectively. Subordinate follicles of wave 2 that had regressed to a 3-mm category (3.0-3.9 mm) or 4-mm category by hour 0 decreased in diameter for at least 48 h before hour 0, whereas follicles that were in the 5-mm or 6-mm categories at hour 0 did not change significantly in diameter during the previous 48 h. About 55% of the follicles that had regressed to the 3-mm and 4-mm categories at hour 0% and 78% of the follicles in the 5-mm and 6-mm categories increased in diameter after gonadotropin treatment, whereas follicles in the control group continued to decrease (regress) in diameter. The follicles for each of the 4 diameter categories were greater (P < 0.05) in diameter 9 h after treatment in the treated group than in the control group. The dominant follicle of wave 1 and the largest subordinate follicle of wave 2 in the treated group also increased in diameter so that diameter was greater (P < 0.05) than in the controls at hour 9. The results demonstrated that subordinate follicles of wave 2 that had decreased in diameter (regressed) for at least 48 h retained the capability to recover as indicated by a diameter increase when exposed to a gonadotropin product.

Conversion of intraovarian patterns from preovulation to postovulation based on location of dominant follicle and corpus luteum in heifers.

The conversion of preovulatory intraovarian patterns based on location of the preovulatory follicle (PF) and the associated corpus luteum (cl) to postovulatory patterns based on location of the future and established dominant follicle (DF) and corpus luteum (CL) was studied daily in 26 heifers from Days -5 to 6 (Day 0 = ovulation). The two ipsilateral preovulatory patterns were PF-cl and devoid (neither PF nor cl), and the two contralateral patterns were PF and cl. The postovulatory patterns were DF-CL, devoid, DF, and CL. For the contralateral preovulatory relationships, a conversion from PF to DF-CL and the accompanying conversion from cl to devoid occurred most frequently (17 of 18 conversions, 94%). For the ipsilateral preovulatory relationships, a conversion from PF-cl to CL and from devoid to DF occurred most frequently (6 of 8, 75%). Number of 2-mm follicles during preovulation was greatest (P < 0.05) for the devoid and PF patterns, and number of 6-mm follicles during postovulation was greatest (P < 0.05) for the DF-CL and DF patterns. Blood flow resistance at a color Doppler signal in the ovarian pedicle indicated increasing ovarian perfusion over days in the PF to DF-CL and devoid to DF conversions and decreasing perfusion in the PF-cl to CL and cl to devoid conversions. In addition to formation of the CL from the PF, it was interpreted that the conversion of patterns involved number of newly emerging 2-mm follicles per ovary before ovulation and a continuation of the preovulatory angioarchitecture into postovulation. Results supported the novel hypothesis that the four preovulatory intraovarian patterns determine the frequency of the four postovulatory patterns.

Intraovarian factors associated with switching of a future dominant follicle to a subordinate follicle during induced luteolysis in heifers.

The factors involved in the switching of a future dominant follicle (DF) to subordinate status were studied (n = 42) by induction of luteolysis with PGF2α (hour 0) when the largest follicle (F1) in follicular wave 2 was 7.0 or 8.5 mm. Combined for 7.0- and 8.5-mm groups, the frequency of switching was greater (P < 0.01) when F1 and CL were ipsilateral (10 of 28, 36%) than when contralateral (0 of 14). The frequency of switching in the ipsilateral relationship was greater (P < 0.002) when F1 and CL were adjacent (<3 mm apart; 10 of 17) than when separated (0 of 11). The difference in diameter between F1 and F2 was less (P < 0.005) when switching occurred (0.4 ± 0.1 mm) than when switching did not occur (ipsilateral, 1.3 ± 0.2 mm; contralateral, 1.1 ± 0.2 mm). Treatment at hour 0 when F1 was 7.0 mm and ipsilateral to the CL resulted in smaller diameter (P < 0.001) of F1 at hour 12 (7.6 ± 0.2 mm) than when treatment was not given (8.3 ± 0.1 mm). The hypotheses were supported that (1) switching from a future DF to a future subordinate is functionally related to luteolysis, and (2) factors involved in switching include an ipsilateral relationship between the largest follicle and CL and close intraovarian proximity of the follicle to the regressing CL. It is proposed that switching of the future DF to subordinate status during spontaneous luteolysis accounts for the reported greater frequency of the contralateral than ipsilateral relationships between the preovulatory follicle and CL in three-wave interovulatory intervals.

Effect of intraovarian proximity between dominant follicle and corpus luteum on dimensions and blood flow of each structure in heifers.

An intraovarian positive physiologic coupling between the extant CL and the ipsilateral preovulatory follicle (PF) or the future or established postovulatory dominant follicle (DF) was studied in 26 heifers. Ovaries were scanned by ultrasonic imaging from Day 16 (Day 0 = ovulation) of the preovulatory period until Day 6 of the postovulatory period. Hemodynamics of the follicles and CL were assessed by color-Doppler ultrasonography. When the PF and CL were ipsilateral compared with contralateral, blood-flow resistance in wall of the PF was lower (P < 0.04) on Days -2 and -1, and percentage blood-flow signals in the CL approached being greater (P < 0.08) on Days -4 to -1. During the postovulatory period, percentage of DF wall with blood-flow signals (44.1 ± 1.2% vs. 31.4 ± 2.8%) and percentage of CL with blood-flow signals (51.8 ± 1.2% vs. 42.5 ± 3.1%) were each greater (P < 0.05) when the two ipsilateral structures were adjacent (distance between antrum and CL wall, ≤ 3 mm) than when separated. On Day 0, the distance between follicle and ipsilateral CL was less (P < 0.02) for the future DF than for the future largest subordinate. Growth rate between Days 0 and 2 averaged over all growing follicles was greater (P < 0.01) when the follicles were ≤3 mm from the CL (1.1 ± 0.1 mm/day) than when farther from the CL (0.9 ± 0.1 mm/day). Results supported the hypotheses that (1) a positive intraovarian coupling occurs between the PF or postovulatory DF and the extant CL and (2) the coupling is enhanced when the ipsilateral DF and CL are in close proximity.

Blood flow to follicles and CL during development of the periovulatory follicular wave in heifers.

The hemodynamics of the developing CL and the future dominant follicle (DF) was studied in 22 heifers during wave 1 on Days 0 to 5 (Day 0 = ovulation). Color-Doppler ultrasonography was used to determine the resistance index (RI) at the most prominent Doppler signal in an ovarian arterial branch before entry into the ovary; a decrease in RI indicates a downstream increase in vascular perfusion. The RI for each of four intraovarian patterns averaged over days was different (P < 0.05) from each of the other patterns as follows: DF-CL (DF and CL in the same ovary), 0.52 ± 0.02; CL alone, 0.60 ± 0.01; DF alone, 0.67 ± 0.01; neither DF nor CL, 0.78 ± 0.01. The differences in RI among intraovarian patterns began on Day 0 or 1, indicating that the extent of vascular perfusion on Days 0 to 5 for the various patterns may have been influenced by events that occurred before ovulation. The percentage of the DF wall with color-flow signals was greater (P < 0.05) in the DF-CL pattern than in the DF pattern on each of Days 2 to 5 and was greater (P < 0.0001) in the DF-CL pattern when the DF was adjacent to the CL (40.2 ± 2.0%) than when separated (24.5 ± 1.9%). Dimensions of DF (P < 0.01) and CL (P < 0.02) were greater when adjacent to each other. The results supported the hypotheses for wave 1 that (1) vascular perfusion is greater for the DF-CL intraovarian pattern than for the DF or CL pattern and (2) the extent of blood-flow Doppler signals in the wall of the developing DF is greater for the DF-CL pattern than for the DF pattern. Our preferred interpretation is that a change in vascular perfusion of the CL is accompanied by a similar change in perfusion of the DF when the two structures are in the same ovary especially adjacent.

Intraovarian effect of dominant follicle and corpus luteum on number of follicles during a follicular wave in heifers.

The intraovarian relationships among dominant follicle (DF), corpus luteum (CL), and number of follicles between Days 0 to 5 (Day 0 = ovulation) in wave 1 (n = 65 waves) and Days 9 to 13 in wave 2 (n = 62) were analyzed in separate experiments in Bos taurus heifers. Ovaries were grouped into intraovarian patterns of DF-CL, DF alone, CL alone, and neither DF nor CL. In wave 1, the pattern frequencies of DF-CL or neither DF nor CL (34% each) were greater (P < 0.0004) than for DF alone or CL alone (16% each). The number of growing follicles ≥5.0 mm, was greater (P < 0.0001) in ovaries with the DF, even when the DF was removed from the tally (P < 0.03). In a factorial analysis of wave 1, there was a positive main effect of DF (3.9 ± 0.2 vs. 2.2 ± 0.2 follicles; P < 0.0001), but the main effect of CL and the interaction of DF and CL were not significant. In a factorial analysis of wave 2, there were more (P < 0.0001) follicles greater than 6 mm in ovaries with a DF when the DF was included and an approaching difference (P < 0.09) when the DF was excluded. The main effect of CL and the interaction of DF and CL were not significant. The hypothesis that both the DF and CL have a positive intraovarian effect on number of follicles in waves 1 and 2 was only partly supported; the DF, but not the CL, had an effect in the factorial analyses. Previous reports in cattle and sheep of a positive intraovarian effect of CL on number of follicles are questionable in that location of the DF was not considered.

Relationship between days of the luteolytic period and locations of the preovulatory follicle and CL in interovulatory intervals with two or three follicular waves in heifers.

Diameter of follicles was determined every 12 hours and progesterone (P4), FSH, and LH concentrations were determined every 6 hours from Day 12 (Day 0 = ovulation) to the ovulation at the end of the interovulatory interval (IOI). Groups were assigned on the basis of an ipsilateral (Ipsi) versus contralateral (Contra) relationship between the preovulatory follicle and CL and two follicular waves (2W) versus three waves (3W) per IOI. Numbers of IOIs were Ipsi-2W (n = 6), Ipsi-3W (n = 6), and Contra-3W (n = 8). Normalization to the end of luteolysis (day that P4 was closest to 1.0 ng/mL) indicated for the first time that concentrations of P4 and FSH were greater (P < 0.05) in 3W IOIs than in 2W IOIs for the 3 days before the beginning of a P4 decrease. The beginning of a P4 decrease occurred about 5 days and 6 hours after emergence of the preovulatory wave at 6 mm in 2W and 3W IOIs, respectively. On the day of diameter deviation between the future dominant and largest subordinate follicles in wave 3 of 3W IOIs, the future dominant follicle had the following characteristics: (1) distribution of diameters differed (P < 0.01) from unimodality; (2) diameter was greater (P < 0.05) in the Contra-3W group (9.8 ± 0.4 mm) than in the Ipsi-3W group (8.8 ± 0.3 mm); (3) diameter was similar to the diameter at the beginning of the P4 decrease (9.6 ± 0.9 mm); and (4) diameter was as small or smaller than diameter of the largest subordinate in seven of 14 heifers compared with zero of seven heifers in wave 2 of 2W IOIs. The differences involving deviation may be related to a reported greater frequency of the Contra-3W group than Ipsi-3W group. Results supported the hypothesis that emergence of the ovulatory wave occurs well before the beginning of luteolysis in 2W IOIs and near the beginning of luteolysis in 3W IOIs.

Complex interrelationships among CL, preovulatory follicle, number of follicular waves, and right or left ovaries in heifers.

The diameter of the dominant follicle (DF) of wave 1 was studied on Days 9 to 17 (Day 0 = ovulation) in a survey of the ipsilateral and contralateral relationships between the location of the DF and CL, and number of follicular waves per interovulatory interval (IOI). For contralateral relationships, regardless of number of waves the diameter of the DF of wave 1 decreased (P < 0.03) between Days 11 and 13 when referenced to the follicle-CL relationship of wave 1 and decreased (P < 0.008) between Days 9 and 11 when referenced to the preovulatory follicle (PF)-CL relationship. For wave 2 in two-wave IOIs, the CL ovary of ipsilateral relationships had more (P < 0.05) follicles that reached at least 6 mm than the non-CL ovary. In three-wave IOIs, frequency of IOIs with the DF in the CL ovary was greater (P < 0.02) for wave 2 than for wave 3. In wave 3, the preovulatory and the largest subordinate follicles were located more frequently (P < 0.005) in the contralateral ovary. Ovulation in two-wave IOIs occurred more frequently (P < 0.0009) from the right ovary. In three-wave IOIs with a contralateral relationship ovulation occurred more frequently (P < 0.003) from the left ovary; a negative intraovarian effect of the CL on location of the PF may account for more ovulations from the left ovary and a reported greater frequency of the contralateral relationship. The hypothesis was supported that the ipsilateral versus contralateral relationship between the PF and CL is affected by the DF-CL relationship during the previous follicular waves and by the number and identity of waves per IOI.

Minor FSH surge, minor follicular wave, and resurgence of preovulatory follicle several days before ovulation in heifers.

Blood samples were collected and follicle diameters were determined daily beginning on Day 12 (Day 0 = ovulation) in 35 interovulatory intervals (IOIs) in heifers. A minor follicular wave with maximal diameter (6.0 ± 0.3 mm) on Day -4 was detected in six of seven IOIs that were scanned for follicles 4 mm or greater. The number of IOIs with a CV-identified minor FSH surge toward the end of the IOI was greater (P < 0.03) in two-wave IOIs (10/17) than in three-wave IOIs (4/18). The 17 two-wave IOIs were used for study of the temporal relationships among preovulatory follicle, FSH, LH, and estradiol. Daily growth rate of the preovulatory follicle was maximum on Days -11 to -7, minimum (P < 0.05) on Days -7 to -4, and increased (resurged, P < 0.05) on Days -4 to -3. A transient increase in FSH was maximum on mean Day -4, and the peak of a minor FSH surge occurred on Day -4.5 ± 0.2. Concentration of LH and estradiol increased between Days -5 and -4. Results demonstrated resurgence of the preovulatory follicle apparently for the first time in any species. Resurgence seemed more related temporally to the minor FSH surge than to the LH increase, but further study is needed. Results supported the novel hypotheses that a minor FSH surge near the end of the IOI is temporally associated with (1) the emergence of a minor follicular wave and (2) the resurgence in growth rate of the preovulatory follicle.

Hormone concentrations temporally associated with contralateral and ipsilateral relationships between the CL and preovulatory follicle during the third follicular wave in heifers.

Concentrations of circulating hormones after Day 14 (Day 0 = ovulation) were determined daily in 87 interovulatory intervals (IOIs) in heifers. The IOIs were grouped into four permutations according to an ipsilateral (Ipsi) or contralateral (Contra) relationship between the CL and the preovulatory follicle and two (2W) or three (3W) follicular waves per IOI. The number of IOIs per group differed (P < 0.005) from equality among the Ipsi-2W (n = 27), Contra-2W (n = 31), Ipsi-3W (n = 9), and Contra-3W (n = 20) groups. A continuous decrease in progesterone (luteolysis) began later (P < 0.05) in the Contra-3W group (Day 18.0 ± 0.4) than in each of the Ipsi-2W (15.4 ± 0.2), Contra-2W (15.6 ± 0.2), and Ipsi-3W (16.2 ± 0.5) groups. Concentrations of LH and estradiol began to increase near the beginning of luteolysis in each group. A minor FSH surge that did not stimulate a major follicular wave developed in about 50% of the IOIs in each group, except that none were detected in the Ipsi-3W group. The minor FSH surge reached a peak about 4 days before ovulation and several days after wave 3 had emerged. The hypothesis that luteolysis begins earliest in two-wave IOIs, intermediate in three-wave IOIs with an ipsilateral CL/follicle relationship, and latest in three-wave IOIs with a contralateral relationship was supported. The hypothesis that a minor FSH surge occurs most frequently in association with three follicular waves was not supported.

Two-way coupling between FSH and the dominant follicle in heifers.

Follicular Wave 1 and 2 and the associated FSH Surge 1 and 2 were used to designate the first two waves and surges of the interovulatory interval in two experiments in heifers. In experiment 1, a group with early (group E, N = 9) and late (group L, N = 5) development of the dominant follicle of Wave 1 were used as natural models to study FSH/follicle coupling. The day of wave emergence and the day of deviation in diameters between the two largest follicles were not different between groups. Emergence of Wave 2 and maximal FSH concentration in Surge 2 was approximately 1 day later (P < 0.03) in group L. Diameter of the dominant follicle of wave 1 (13.8 ± 0.3 mm vs. 12.0 ± 0.3 mm) and FSH concentrations in Surge 2 (0.29 ± 0.02 ng/mL vs. 0.21 ± 0.03 ng/mL) were first greater (P < 0.05) in group E than in group L at 4 and 5 days, respectively, after wave emergence. In experiment 2, treatment with estradiol (N = 8) when the dominant follicle of Wave 1 was ≥ 11 mm (Hour 0) resulted in a decrease (P < 0.02) in FSH and slower (P < 0.05) growth rate of the follicle between Hours 0 and 4. Results supported the following hypotheses: (1) the FSH surge that stimulates emergence of a follicular wave is associated with final growth of the dominant follicle of the previous anovulatory wave; and (2) suppression of FSH Surge 2 when the dominant follicle of Wave 1 is ≥ 11 mm is associated with a decrease in diameter. It is concluded for the first time that two-way FSH/follicle coupling in heifers continues during final growth of the dominant follicle of Wave 1 and that Surge 2 is the FSH source.

Endocrinology of number of follicular waves per estrous cycle and contralateral or ipsilateral relationship between corpus luteum and preovulatory follicle in heifers.

A 3-d extension of the luteal phase occurs in interovulatory intervals (IOIs) with a contralateral relationship between the corpus luteum (CL) and preovulatory follicle with 3 follicular waves (Contra-3W group). Concentrations of FSH, progesterone, LH, and estradiol-17ß for the ipsilateral versus contralateral CL and/or follicle relationship and 2 versus 3 waves per IOI were studied in 14 heifers. Follicular waves and FSH surges were designated 1, 2, or 3, according to order of occurrence in the IOI. The day (day 0 = ovulation) of the FSH peak in surge 2 occurred earlier (P < 0.02) in 3-wave IOIs (day 6.3 ± 0.5) than in 2-wave IOIs (day 8.5 ± 0.5). Mean FSH was higher in 3-wave than in 2-wave IOI on 82% of the days in the IOI. Repeatability or individuality in FSH concentration was indicated by a correlation (r = 0.54, P < 0.04) in FSH concentrations between ovulations at the beginning and at the end of the IOI. Concentrations of LH and estradiol increased (P < 0.05) near the beginning of the luteolytic period in 2-wave IOI regardless of the CL and/or follicle relationship. In the Contra-3W group, LH and estradiol remained at basal concentrations concurrently with FSH surge 3 and extension of the luteal phase. The hypotheses were supported that FSH surge 2 occurs earlier in 3-wave IOIs than in 2-wave IOIs and that the development of 3-wave IOIs occurs in individuals with greater FSH concentrations. Extension of the luteal phase in the Contra-3W group was temporally associated with lower concentrations of LH and estradiol.

Volume-dependent glutamate permeation depends on transmembrane ionic strength and extracellular Cl-.

Many mammalian cells regulate their volume by the osmotic movement of water directed by anion and cation flux. Ubiquitous volume-dependent anion currents permit cells to recover volume after swelling in response to a hypotonic environment. This study addressed competition between glutamate (Glu) and Cl(-) permeation in volume-activated anion currents in order to provide insight into the ionic requirements for volume regulation, volume-dependent anion channel activity and to the architecture of the channel pore. The effect of changing the intracellular molar fraction (MF) of Glu and Cl(-) on conductance and relative anion permeability was evaluated as a function of the extracellular permeant anion and/or the ionic strength. Relative permeability of Glu to Cl(-) was determined by measuring reversal potentials under defined ionic conditions. Under conditions with high (150 m M) or low (50 m M) ionic strength solutions on both sides of the membrane, Cl(-) was always more permeable than Glu. When a transmembrane ionic strength gradient (150 m M extracellular: 50 m M intracellular) was set to drive water into the cell, and in the presence of extracellular Cl(-), Glu became up to 16-fold more permeable than Cl(-). Replacement of extracellular Cl(-) with Glu abolished this effect. These results indicate that it is possible for Glu to move into the extracellular environment during volume-regulatory events and they support the emerging role of glutamate as a modulator of anion channel activity.

Modulation of volume regulated anion current by I(Cln).

I(Cln), a cytosolic protein associated with a nucleotide-sensitive chloride current, may be involved in the regulation of a volume-regulated anion current (VRAC) associated with hypotonic cell swelling. We have determined the nucleic acid sequences of I(Cln) from human tsA201a, colonic (T84) and myeloma (RPMI 8826) cell lines. The amino acid sequences are highly homologous (>/=99%) to each other but less homologous to I(Cln) protein from other species. Using the whole-cell patch clamp technique, we examined the effect of I(Cln) protein expression levels on VRAC properties during a hyposmotic challenge. Overexpression of T84 or RPMI 8226-derived I(Cln) protein in tsA201a cells results in a more than 9-fold increase in the rate of VRAC activation over control values, while having no effect on VRAC inactivation properties. Underexpression of endogenous I(Cln) protein in tsA201a cells using antisense oligonucleotides results in a more than 180-fold decrease in VRAC activation rate as compared to control values. These results indicate that I(Cln) protein expression modulates VRAC activation but not inactivation.

Evidence for altered ion transport in Saccharomyces cerevisiae overexpressing human MDR 1 protein.

Recently [Hoffman, M. M., and Roepe, P. D. (1997) Biochemistry 36, 11153-11168] we presented evidence for a novel Na+- and Cl--dependent H+ transport process in LR73/hu MDR 1 CHO transfectants that likely explains pHi, volume, and membrane potential changes in eukaryotic cells overexpressing the hu MDR 1 protein. To further explore this process, we have overexpressed human MDR 1 protein in yeast strain 9.3 following a combination of approaches used previously [Kuchler, K., and Thorner, J. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 2302-2306; Ruetz, S., et al. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 11588-11592]. Thus, a truncated hu MDR 1 cDNA was cloned behind a tandem array of sterile 6 (Ste6) and alchohol dehydrogenase (Adh) promoters to create the yeast expression vector pFF1. Valinomycin resistance of intact cells and Western blot analysis with purified yeast plasma membranes confirmed the overexpression of full length, functional, and properly localized hu MDR 1 protein in independently isolated 9.3/pFF1 colonies. Interestingly, relative valinomycin resistance and growth of the 9.3/hu MDR 1 strains are found to strongly depend on the ionic composition of the growth medium. Atomic absorption reveals significant differences in intracellular K+ for 9.3/hu MDR 1 versus control yeast. Transport assays using [3H]tetraphenylphosphonium ([3H]TPP+) reveal perturbations in membrane potential for 9.3/hu MDR 1 yeast that are stimulated by KCl and alkaline pHex. ATPase activity of purified plasma membrane fractions from yeast strains and LR73/hu MDR 1 CHO transfectants constructed previously [Hoffman, M. M., et al. (1996) J. Gen. Physiol. 108, 295-313] was compared. MDR 1 ATPase activity exhibits a higher pH optimum and different salt dependencies, relative to yeast H+ ATPase. Inside-out plasma membrane vesicles (ISOV) fabricated from 9.3/hu MDR 1 and control strains were analyzed for formation of H+ gradients +/- verapamil. Similar pharmacologic profiles are found for verapamil stimulation of MDR 1 ATPase activity and H+ pumping in 9.3/hu MDR 1 ISOV. In sum, these experiments strongly support the notion that hu MDR 1 catalyzes H+ transport in some fashion and lowers membrane potential in yeast when K+ contributes strongly to that potential. In the accompanying paper [Santai, C. T., Fritz, F., and Roepe, P. D. (1999) Biochemistry 38, XXXX-XXXX] the effects of ion gradients on H+ transport by hu MDR 1 are examined.

Analysis of ion transport perturbations caused by hu MDR 1 protein overexpression.

In previous work [Luz et al. (1994) Biochemistry 33, 7239-7249; Roepe et al. (1994) Biochemistry 33, 11008-11015] we measured changes in Cl- and HCO3--dependent pHi regulation for LR73 Chinese hamster ovary fibroblasts overexpressing mu MDR 1 protein. However, only one clonal cell line overexpressing the protein but not previously exposed to chemotherapeutic drug (i.e., a "true" transfectant) was examined, since very few MDR cell lines of this nature have been constructed. Recently [Hoffman et al. (1996) J. Gen. Physiol. 108, 295-313] we derived a series of true LR73/hu MDR 1 transfectants that are valuable for defining the MDR phenotype mediated by MDR protein alone, without the additional complexities introduced by exposing cells to chemotherapeutic drugs. Several independently derived clones from these and additional transfection experiments exhibit expression of MDR protein that is higher than that found in other true transfectants, and that is similar to the highest level of overexpression yet recorded for drug selected MDR cells. We examined altered Cl--dependent pHi regulation for these clones using improved single-cell photometry (SCP) techniques. Short-term isotonic Cl- substitution experiments performed in the presence of CO2/HCO3- reveal that mild overexpression of hu MDR 1 protein alters anion exchange (Cl-/HCO3- exchange or AE) for LR73 cells, as expected on the basis of previous work [Luz et al. (1994) Biochemistry 33, 7239-7249]. Interestingly, we now find that several independently selected high-level MDR 1 overexpressing clones acidify quite extensively upon isotonic exchange of Cl- and then rapidly alkalinize upon restoring normal [Cl-]. These data suggest that MDR protein may effectively compete against AE. The MDR protein effect is not dependent on HCO3-/CO2 or K+, is partially inhibited by verapamil, is completely inhibited by substituting K+ or N-methylglucamine (NMG+) for Na+ in the SCP perfusate but is not affected by 100 microM levels of amiloride, bumetanide, chlorothiazide, or stilbene. ATP depletion inhibits the MDR 1 effect. We are unable to restore normal AE activity for the MDR clones via manipulation of Cl- or HCO3- gradients. We thus suggest that MDR protein overexpression provides a novel Na+- and Cl--dependent pathway for transmembrane H+ transport. From analysis of ion dependency and inhibitor sensitivities, we conclude the transport is not via altered regulation of any known K+/H+, Na+/H+, or Cl-/HCO3- antiporters, Na+:K+:2Cl-, Na+:K+:2HCO3-, K+:HCO3-, or Na+:HCO3- co-transporters, or any combination of these. Thus, it appears to represent a novel ATP and Na+-dependent Cl-/H+ antiport process that (1) may be directly mediated by the MDR protein, (2) may represent the modulation of one or more currently unidentified ion transport proteins by MDR protein, (3) may be due to some combination of direct ion transport and regulation of ion transport, or (4) may represent unusual passive H+ movement in response to a novel Cl--dependent electrical perturbation that occurs during our Cl- substitution protocol. The results have important implications for understanding drug resistance mediated by MDR 1 overexpression, as well as the physiologic function of endogenously expressed MDR protein.

Altered pHi regulation in 3T3/CFTR clones and their chemotherapeutic drug-selected derivatives.

Recently (L. Y. Wei, M. J. Stutts, M. M. Hoffman, and P. D. Roepe. Biophys. J. 69: 883-895, 1996), 3T3 cells overexpressing the cystic fibrosis transmembrane conductance regulator (CFTR) were found to exhibit chemotherapeutic drug resistance and other traits of multidrug resistant (MDR) cells. In the present work, NIH 3T3/CFTR clones were selected with either doxorubicin or vincristine in incremental fashion to generate series of stable MDR cell lines that exhibit increasing levels of drug resistance. Thus C3D6 (grown in the presence of 600 nM doxorubicin) was selected from C3D4 (grown in the presence of 400 nM doxorubicin), which was selected from C3D1 (grown in the presence of 100 nM doxorubicin), which was in turn selected from the original 3T3/CFTR clone C3 (M. J. Stutts, S. E. Gabriel, J. C. Olsen, J. T. Gatzy, T. L. O'Connell, E. M. Price, and R. C. Boucher. J. Biol. Chem. 268: 20653-20658, 1993), which was not grown in the presence of chemotherapeutic drug. A similar series was generated via selection with vincristine. In both series, as well as series derived from a different CFTR clone, initial low-level drug selection increases CFTR expression without promoting MDR 1 or multidrug resistance-associated protein expression. On continued selection at higher drug concentrations, CFTR mRNA levels decrease while MDR 1 mRNA levels concomitantly increase. At each incremental step of selection, intracellular pH (pHi) increases (e.g., pHi of C3D6 > C3D4 > C3D1 > C3). Cl-/HCO3- exchange activity is significantly reduced in the drug-selected derivatives overexpressing MDR 1 but not the parental CFTR clones. The apparent set point of Na+/H+ exchange activity is significantly lower for the non-drug-selected 3T3/CFTR clones, relative to controls, but it increases on initial selection with chemotherapeutic drug. Overexpression of MDR 1 in the higher-level selectants does not appear to further perturb apparent Na+/H+ exchange. These data further describe how CFTR and MDR proteins may affect pHi regulation.